Biologicals and their use in plants

ABSTRACT

Entomopathogenic fungal strains, compositions, and methods and compositions of producing and using the strains for reducing overall insect damage.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to International Application NumberPCT/US2016/055913, filed Oct. 7, 2016, which claims priority to UnitedStates Provisional Application No. 62/336,902, filed May 16, 2016, whichare hereby incorporated herein in their entireties by reference.

FIELD

Entomopathogenic fungal strains, compositions, and methods andcompositions for producing and using entomopathogenic fungal strains forreducing overall insect damage.

BACKGROUND

There has been a long felt need for environmentally friendlycompositions and methods for controlling or eradicating insect pests ofagricultural significance, i.e., methods that are selective,environmentally inert, non-persistent, and biodegradable, and that fitwell into insect pest management schemes. Furthermore, there is a needto produce such compositions on a large scale for commercial use.

SUMMARY

In an embodiment, a method of producing a fungal entomopathogenicproduct in liquid culture is disclosed. In an embodiment, a method ofproducing an entomopathogenic product comprising an entomopathogenicfungal strains selected from the group consisting of Metarhiziumanisopliae 15013-1 (NRRL 67073), Metarhizium robertsii 23013-3 (NRRL67075), Metarhizium anisopliae 3213-1 (NRRL 67074), or any combinationsthereof is disclosed. In another embodiment, a method of producingspores in liquid culture is disclosed. In an embodiment, a method ofproducing microslerotia in liquid culture is disclosed. In anotherembodiment, a method of producing conidiospores or blastospores inliquid culture is disclosed. In a further embodiment, a method ofproducing submerged spores in liquid culture is disclosed. In anotherembodiment, a method of producing vegetative mycelium in liquid cultureis disclosed. In a further embodiment, a method comprises obtaining afungal entomopathogenic product from a liquid medium. In anotherembodiment, a composition may be the product of a disclosed method ofproduction.

In an embodiment, a method of producing a fungal entomopathogenicproduct in liquid culture comprising obtaining aerial conidospores of afungal entomopathogen, inoculating the aerial conidiospores into aliquid medium to generate a fungal entomopahogenic seed culture byfermentation, and inoculating the fungal entomopathogen seed cultureinto a liquid medium to generate a fungal entomopahogenic product byfermentation, wherein the liquid medium comprises a first carbon source,a second carbon source, and a nitrogen source is disclosed. In a furtherembodiment, a method comprises obtaining a fungal entomopathogenicproduct from a liquid medium.

In another embodiment, a composition comprises a fungal entomopathogenicproduct, wherein the fungal entomopathogenic product has insecticidalactivity. A fermentation product may comprise spent media or broth, afungal entomopathogen, a spore, a mycelium, a microsclerotia, aconidiospore, a blastospore, a submerged spore, a vegetative mycelium,or any other substantially pure component of the fermentation media orlysate.

In another embodiment, a method of producing a fungal entomopathogen ina liquid culture using a carbon source and a nitrogen source isdisclosed. In another embodiment, a method of producing a fungalentomopathogen in a liquid culture using a first carbon source, a secondcarbon source, and a nitrogen source is disclosed. In anotherembodiment, a method of producing a fungal entomopathogen in a liquidculture using two or more carbon sources and a nitrogen source isdisclosed. In an embodiment, a carbon source may include a glucose, afructose, a galactose, a sorbitol, a sorbose, a sucrose, an arabinose, amaltodextrin, a ribose, or a xylose carbon source and combinationsthereof. In another embodiment, a second carbon source comprises afructose, a galactose, a sorbitol, a sorbose, a sucrose, an arabinose, amaltodextrin, a ribose, or a xylose carbon source and combinationsthereof. In a further embodiment, a second carbon source creates anon-optimal or stress condition that changes the physiological state ofa fungal entomopathogen. In an embodiment, a first carbon source is in alimiting concentration to facilitate a non-optimal or stress conditionof a fungal entomopathogen. In an embodiment, a first carbon sourcecomprises 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, or 40% of the totalcarbon source amount (weight/volume). In an embodiment, a second carbonsource is not in a limiting concentration.

In another embodiment, a method of producing a fungal entomopathogen ina liquid culture using a carbon source and a nitrogen source andcontrolling a fermentation parameter, wherein controlling thefermentation parameter creates a non-optimal or stress condition thatchanges a physiological state of a fungal entomopathogen is disclosed.In an embodiment a fermentation parameter may include a pH level, acarbon dioxide evolution rate, a dissolved oxygen percentage, anagitation profile, a sugar feed rate, or any other measured parameter ofa fermentation of a fungal entomopathogen that may create a non-optimalor stress condition resulting in a change of a physiological state of afungal entomopathogen. In a further embodiment, a method of producing afungal entomopathogen in a liquid culture using a first carbon source, asecond carbon source, and a nitrogen source, wherein the second carbonsource creates a non-optimal or stress condition that changes aphysiological state of a fungal entomopathogen is disclosed.

In an embodiment, a medium for producing a fungal entomopathogenicproduct comprising a first carbon source, a second carbon source, and anitrogen source, wherein the second carbon source creates a non-optimalor stress condition of a fungal entomopathogen is disclosed. In anembodiment, a carbon source may include a glucose, a fructose, agalactose, a sorbitol, a sorbose, a sucrose, an arabinose, amaltodextrin, a ribose, or a xylose carbon source and combinationsthereof. In another embodiment, a second carbon source comprises afructose, a galactose, a sorbitol, a sorbose, a sucrose, an arabinose, amaltodextrin, a ribose, or a xylose and combinations thereof. In anembodiment, a first carbon source is in a limiting concentration tofacilitate a non-optimal stress condition of a fungal entomopathogen. Inan embodiment, a first carbon source comprises 1, 2, 3, 4, 5, 10, 15,20, 25, 30, 35, or 40% of the total carbon source amount(weight/volume). In an embodiment, a second carbon source is not in alimiting concentration.

DESCRIPTION OF THE DRAWINGS

FIG. 1. Production of Metarhizium anisopliae strain 15013-1 fungalpropagules during liquid fermentation at shake flask scale; A.Microsclerotia (MS) production after 3, 5, and 7 days of liquidfermentation in soy 10C (25% glucose+75% fructose):1N medium at 300 rpmand 28° C., and B. Submerged spore (SS) production after 3, 5, and 7days of liquid fermentation in soy 10C (25% glucose+75% fructose): 1Nmedium at 300 rpm and 28° C.

FIG. 2. Fermentation process conditions during production of Metarhiziumanisopliae strain 15013-1 fungal propagules at 2 L benchtop scale; A.CER profile, and B. DO % profile.

FIG. 3. Fermentation process conditions during production of Metarhiziumanisopliae strain 15013-1 fungal propagules at 2 L benchtop scale; A.Ammonia concentration profile, and B. pH profile.

FIG. 4. Production of Metarhizium anisopliae strain 15013-1 biomass andfungal propagules during fermentation at 2 L benchtop scale; A. Dry CellWeight (DCW) profile, and B. Microsclerotia (MS) production profile.

FIG. 5. Submerged spore (SS) production profile of Metarhiziumanisopliae strain 15013-1 during fermentation at 2 L benchtop scale.

FIG. 6. Fermentation process conditions during production of Metarhiziumanisopliae strain 15013-1 fungal propagules without feeding; A. CERprofile, and B. DO % profile.

FIG. 7. Fermentation process conditions during production of Metarhiziumanisopliae strain 15013-1 fungal propagules without feeding; A. Ammoniaconcentration profile, and B. pH profile.

FIG. 8. Production of Metarhizium anisopliae strain 15013-1 biomass andfungal propagules during fermentation without feeding; A. Dry CellWeight (DCW) profile, and B. Microsclerotia (MS) production profile.

FIG. 9. Submerged spore (SS) production profile of Metarhiziumanisopliae strain 15013-1 during fermentation without feeding.

FIG. 10. Fermentation process conditions during production ofMetarhizium anisopliae strain 15013-1 fungal propagules with feeding; A.CER profile, and B. DO % profile.

FIG. 11. Fermentation process conditions during production ofMetarhizium anisopliae strain 15013-1 fungal propagules with feeding; A.Ammonia concentration profile, and B. smoothed specific sugar feed rateprofile.

FIG. 12. Fermentation process conditions during production ofMetarhizium anisopliae strain 15013-1 fungal propagules with feeding at;A. pH profile, and B. Dry Cell Weight (DCW) profile.

FIG. 13. Production of Metarhizium anisopliae strain 15013-1 fungalpropagules during fermentation with feeding; A. Microsclerotia (MS)production profile, and B. Submerged spore (SS) production profile.

FIG. 14. Fermentation process conditions during production of submergedspore (SS)-enriched Metarhizium anisopliae strain 15013-1 fungalpropagules; A. CER profile, and B. DO % profile.

FIG. 15. Fermentation process conditions during production ofSS-enriched Metarhizium anisopliae strain 15013-1 fungal propagules; A.Ammonia concentration profile, and B. pH profile.

FIG. 16. Production of Metarhizium anisopliae strain 15013-1 biomass andfungal propagules during fermentation enriched for submerged spore (SS)production; A. Dry Cell Weight (DCW) profile, and B. Microsclerotia (MS)production profile.

FIG. 17. Submerged spore (SS) production profile of Metarhiziumanisopliae strain 15013-1 during fermentation enriched for SSproduction.

DETAILED DESCRIPTION

The embodiments of the invention are not limited by the exemplarymethods and materials disclosed, and any methods and materials similaror equivalent to those described can be used in the practice or testingof embodiments of this invention. Numeric ranges are inclusive of thenumbers defining the range.

The headings provided are not limitations of the various embodiments ofthis invention, which can be had by reference to the specification.

Definitions of terms appear throughout the specification. It is to beunderstood the embodiments of the invention are not limited toparticular embodiments described, and additional embodiments may vary.It is also to be understood that the terminology used is to describe theembodiments only, and is not intended to be limiting, since the scope ofthe embodiments of the invention will be limited only by the appendedclaims and equivalents thereof.

The article “a” and “an” are used to refer to one or more than one(i.e., to at least one) of the grammatical object of the article. Forexample, “an element” means one or more element.

As used herein, the terms “liquid fermentation” and “fermentation” referto growth of the fungal entomopathogen in liquid medium to producevarious cellular structures. As used herein, the term “seed culture”refers to a liquid culture or solid state fermentation product used tobuild up the biomass required for starting a production culture. As usedherein, the term “biomass” includes all the structures that can beformed by the fungal enotmopathogen in liquid medium. As used herein,the term “dry cell weight” (DCW) refers to grams of dry weight perkilogram of broth, and will be used as a measure of biomassaccumulation. As used herein, the term CER refers to the rate of carbondioxide produced in the bioreactor defined as mmol CO₂/L of broth/h(Buckland et al., 1985 BioTechnology). As used herein, the termfermentation dissolved oxygen (DO %) is expressed in % of airsaturation.

As used herein, the term a “carbon source” refers to any complex orsimple carbohydrate capable of sustaining survival, growth, orproduction of particular fungal structures of a fungal entomopathogen.In an embodiment, a carbon source may be provided in a fermentationthrough a feed. In a further embodiment, the carbon source provided in afeed comprises glucose or fructose. As used herein, a “limitingconcentration” of a carbon source means a main carbon source, glucosefor example, is depleted and the organism must switch to using a secondcarbon source for survival, growth, or production of particular fungalstructures. As used herein, a “nitrogen source” refers to a complexnitrogen source that may comprise carbon, but is not a carbon sourcecapable of sustaining survival, growth, or production of particularfungal structures of a fungal entomopathogen. In an embodiment, anitrogen source comprises soy, defatted meal, protein concentrate,protein isolate, a hydrolysate, yeast or other extract, molasses or acombination thereof.

As used herein, the term “fermentation product” refers to a compositioncomprising a vegetative mycelium, a spore, a conidiospore, ablastospore, an aerial conidiospore, a submerged conidiospore, asubmerged blastospore, and/or a microsclerotia of a fungalentomopathogen that is produced through a fermentation process. In anembodiment, a fermentation product comprises a Metarhizium fungalentomopathogen. In another embodiment, a fermentation product comprisesan entomopathogenic fungal strain selected from the group consisting ofMetarhizium anisopliae 15013-1, Metarhizium robertsii 23013-3,Metarhizium anisopliae 3213-1. A fermentation product may be obtainedfrom a fermentation process using vacuum drying. In an embodiment, afermentation product is capable of surviving or being dessicantresistant for downstream applications or formulations.

In an embodiment of the invention, a method of producing a fungalentomopathogen product in a liquid fermentation is disclosed. In anembodiment, a method consists of first generating aerial conidiosporesof a fungal entomopathogen on an agar media, and then inoculating theaerial conidiospores into a liquid medium to generate a fungalentomopathogen product. In another embodiment, a method consists offirst generating aerial conidiospores of a fungal entomopathogen on asolid substrate, including, but not limited to, an agar media or othersolid media of appropriate composition, followed by inoculating theaerial conidiospores into a liquid medium to generate a fungalentomopathogen seed culture, followed by inoculating the fungalentomopathogen seed culture into a liquid medium to generate a fungalentomopathogen product. In another embodiment, a first fungalentomopathogen seed culture may be used to generate a second fungalentomopathogen seed culture, wherein the second seed culture is used toinoculate into a liquid medium to generate a fungal entomopathogenproduct. A liquid medium used to produce a fungal entomopathogen productmay contain minerals, vitamins, a carbon source and a complex nitrogensource. In another embodiment, a nitrogen source is a complex sourcethat comprises carbon, but is not a carbon source. In an embodiment, amethod to produce a fungal entomopathogen product comprising a spore, avegetative mycelium, a submerged spore, and/or a microsclerotia isdisclosed. In an embodiment, a composition comprises a fermentationproduct of a fungal entomopathogen from a liquid fermentation. Afermentation product may be vacuum dried for use to control plantpathogens, pests, or insects.

In an embodiment of the invention, a method of producing a fungalentomopathogen product in a liquid fermentation, wherein the liquidfermentation comprises a liquid medium comprising minerals, vitamins, acarbon source, and a nitrogen source is disclosed. In an embodiment, amethod of producing a fungal entomopathogen in a liquid culture using acarbon source and a nitrogen source is disclosed. In another embodiment,t a method of producing a fungal entomopathogen in a liquid cultureusing two carbon sources and a nitrogen source is disclosed. In anotherembodiment, a method of producing a fungal entomopathogen in a liquidculture using two or more carbon sources and a nitrogen source isdisclosed. In an embodiment, a carbon source is glucose. In anotherembodiment, a carbon source comprises a fructose, a galactose, asorbitol, a sorbose, a sucrose, an arabinose, a maltodextrin, a ribose,or a xylose molecule and combinations thereof. In another embodiment, afirst carbon source is in a limiting concentration. In a furtherembodiment, a second carbon source creates a non-optimal or stresscondition that changes a physiological state of a fungal entomopathogen.In another embodiment, a method of producing a fungal entomopathogen ina liquid culture using a carbon source, a first nitrogen source, and asecond nitrogen source, wherein the first nitrogen source is in alimiting concentration is disclosed.

In another embodiment, a method of producing a fungal entomopathogen ina liquid culture using a carbon source and a nitrogen source andcontrolling a fermentation parameter, wherein controlling thefermentation parameter creates a non-optimal or stress condition thatchanges a physiological state of a fungal entomopathogen is disclosed.In an embodiment a fermentation parameter may include a pH level, acarbon dioxide evolution rate, a dissolved oxygen percentage, anagitation profile, a sugar feed rate, or any other measured parameter ofa fermentation of a fungal entomopathogen that may create a non-optimalor stress condition resulting in a change of a physiological state of afungal entomopathogen. Physiological changes (switch to asexual cycle)may occur as a result of imposing stress or non-optimal conditions on afungal emtomopathogen. (See Steyaert et al. (2010), Microbiol. and Gaoet al. (2007) Mycol. Res). In another embodiment, a method of producinga fungal entomopathogen in a liquid culture using at least two carbonsources and a nitrogen source and controlling a fermentation parameter,wherein controlling the fermentation parameter creates a non-optimal orstress condition that results in a change of a physiological state of afungal entomopathogen is disclosed. In an embodiment, obtaining aerialconidospores of a fungal entomopathogen comprises first generatingaerial conidiospores of the fungal entomopathogen on an agar media or asolid state media (Dorta and Arcas (1998), Enzyme Microb. Technol.).

An embodiment of the invention relates to a method for producing afungal entomopathogen, wherein the fungal entomopathogen comprises orconsists of or consists essentially of an entomopathogenic fungal strainselected from the group consisting of: Metarhizium anisopliae 15013-1,Metarhizium robertsii 23013-3, and Metarhizium anisopliae 3213-1. Inanother embodiment, a method comprises, consists of, or consistsessentially of at least two or more entomopathogenic fungal strainsselected from the group consisting of: Metarhizium anisopliae 15013-1,Metarhizium robertsii 23013-3, Metarhizium anisopliae 3213-1.

In an embodiment, a method for producing a fungal entomopathogenicproduct comprises generation of aerial conidiospores (AC) used as aninoculum for liquid cultures or liquid fermentations. Such methodsinclude, but are not limited to, generation of ACs by inoculating afungal entomopathogen strain on large PDA or VM plates and incubating at28° C. for about 2 to 3 weeks; flooding the plates with a solution of0.05% Tween 80; and suspending ACs in the solution by gently scrapingthe surface of the plate culture. In an embodiment, an AC suspension maybe filtered, and the ACs pooled to a high concentration. In a furtherembodiment, an AC concentration may be determined using a hemocytometer,the ACs centrifuged, and the AC pellet re-suspended using a solution of15% glycerol in 0.05% Tween 80. In another embodiment, aerialconidiospores may also be obtained by solid state fermentation (Dortaand Arcas (1998), Enzyme Microb. Technol.).

In an embodiment, producing a fungal entomopathogen product in a liquidculture may comprise media volumes of 50 mL at shake flask fermentationscale, 1 L at 2 L benchtop fermentation scale, and 10 L at bioreactorfermentation scale, or up to 600,000 L fermentation scale. Media forseed or production cultures may comprise components as shown in Tables1, 2 and 3. At shake flask scale media may be inoculated directly withaerial conidiospores (AC) at a final concentration of about 5×10⁶ AC/mL.At benchtop or bioreactor scale media may be inoculated using a seedculture of about 40 mL or 400 mL of seed culture, respectively. A seedculture may be produced to build up biomass for a production culture.Seed cultures may be generated by further incubating a culture fromabout 1 to 7 days at about 28° C., and agitating from about 100 to 300rpm. Upon addition of an inoculum, production cultures may be incubatedfrom about 4 to 7 days at about 16° C. to 32° C. in an orbital shaker atabout 300 rpm at shake flask scale; about 500 to 1200 rpm agitation atbenchtop scale; or with agitation speeds equivalent to the benchtopimpeller tip speed at bioreactor scale. In certain embodiments, watermay be added to reduce the viscosity of a broth during fermentation.Pressure in a fermentation tank may be set at about 0.5 to 1 barg. Incertain embodiments, a 50% (w/w) fructose solution may be fed after aninitial glucose and fructose solution is exhausted. In certainembodiments, a seed or a production culture may have no pH control,one-sided (base addition only) pH control, or two-sided (base and acidaddition) pH control. During a fermentation and/or at the end of afermentation a variety of parameters may be recorded, such as, but notlimited to, microsclerotia (MS) production, submerged spore (SS)production, biomass build-up expressed through grams of dry cell weightper kilogram of broth (DCW), carbon evolution rate (CER), oxygen uptakerate (OUR), dissolved oxygen (DO), ammonia concentration, pH, feed rate,carbon source content, and agitation.

TABLE 1 Vitamins present in all media. Final concentration Vitamins[mg/L] Thiamine•HCl (Vit. B1) 0.5 Riboflavin (Vit. B2) 0.5 CalciumPantonthenate (Vit. 0.5 B5) Nicotinic Acid (Vit. B3) 0.5 Pyridoxamine0.5 Thioctic Acid (Lipoic Acid) 0.5 Folic Acid (Vit. B9) 0.05 D-Biotin(Vit. B7) 0.05 Cobalamin (Vit. B12) 0.05

TABLE 2 Basal salts present in all media. Final concentration Basalsalts [amount/L] KH₂PO₄ 4 g CaCl₂•2H₂O 0.8 g MgSO₄•7H₂O 0.6 g 0.1M CoCl₂1.555 mL 10 g/L MnSO₄•H₂O 1.6 mL 10 g/L ZnSO₄•7H₂O 1.4 mL

TABLE 3 Carbon and nitrogen sources in different liquid media. sourceconcentrations in Soy 10C:1N Soy 10C:1N Soy 10C:1N Soy 10C:1N Soy 10C:1Ndifferent media Soy 10C:1N 25% Glu75% Fru 25% Glu75% Gal 25% Glu75%Sorbitol 25% Glu75% Sorbose 25% Glu75% Suc Soy flour   45 g    45 g   45 g    45 g    45 g    45 g D-Glucose 49.5 g 12.375 g 12.375 g12.375 g 12.375 g 12.375 g D-Fructose 37.125 g D-Galactose 37.125 gD-Sorbitol 37.125 g L-Sorbose 37.125 g Sucrose 37.125 g L-ArabinoseMaltodextrin D-ribose D-xylose source concentrations in Soy 10C:1N Soy10C:1N Soy 10C:1N Soy 10C:1N different media 25% Glu75% Ara 25% Glu75%Mal 25% Glu75% Rib 25% Glu75% Xyl Soy flour    45 g    45 g    45 g   45 g D-Glucose 12.375 g 12.375 g 12.375 g 12.375 g D-FructoseD-Galactose D-Sorbitol L-Sorbose Sucrose L-Arabinose 37.125 gMaltodextrin 37.125 g D-ribose 37.125 g D-xylose 37.125 g *In somecases, soy flour was substituted with other sources of nitrogen, suchas, but not limited to cottonseed flour, yeast extract or Casaminoacids; in some cases the ratio of carbon (C) to nitrogen (N) was 30:1,or 50:1.

In certain embodiments, recovering and formulating a fungalentomopathogen (Metarhizium spp.) product from a liquid culturecomprises cooling and harvesting a fermentation broth. A fermentor maybe rinsed with about 1× to 2× the volume of a fermentation broth, andthe diluted broth pooled with the neat broth. A diluted entomopathogenicfungal material in a fermentation broth may be treated with DE Admix. Atreated fermentation broth may be filtered through a Büchner filter. Afilter cake may be processed immediately, or stored in a cold room untilprocessing. A wet filter cake may be broken up and dried in a vacuumdrier for about 48 h to 5 days. A dried filter cake may be ground tocreate a final entomopathogenic fungal dried powder product.

As used herein, “administer” refers to the action of introducing astrain and/or a composition to an environment for pathogen, pest, orinsect inhibition or to improve plant performance.

As used herein, the term “agrochemically active compounds” refers to anysubstance that is or may be customarily used for treating plantsincluding but not limited to fungicides, bactericides, insecticides,acaricides, nematicides, molluscicides, safeners, plant growthregulators, and plant nutrients, as well as, microorganisms.

As used herein, a composition may be a liquid, a heterogeneous mixture,a homogeneous mixture, a powder, a solution, a dispersion or anycombination thereof.

As used herein, “effective amount” refers to a quantity of anentomopathogenic fungal strain or a composition sufficient to inhibitgrowth of a pathogenic microorganism or to impede the rate of growth. Inanother embodiment, the term “effective amount” refers to a quantity ofan entomopathogenic fungal strain or a composition sufficient to improveplant performance. In another embodiment, the term “effective amount”refers to a quantity of an entomopathogenic fungal strain or acomposition sufficient to control, kill, inhibit, and reduce the number,emergence, or growth of a pathogen, pest, or insect. In anotherembodiment, the term “effective amount” refers to a quantity of anentomopathogenic fungal strain or a composition sufficient to preventdamage from a pathogen, pest, or insect. One skilled in the art willrecognize that an effective amount of an entomopathogenic fungal strainor a composition may not reduce the numbers of pathogens, pests orinsects, but is effective in decreasing damage to plants as a result ofa pathogen, pest or insect. For example, a pesticidally effective amountmay reduce pathogen, pest or insect emergence, or damage to seeds,roots, shoots, or foliage of plants that are treated compared to thosethat are untreated.

As used herein, the term “inhibit” refers to destroy, prevent, reduce,resist, control, decrease, slow or otherwise interfere with the growthor survival of a pathogen, pest, or insect when compared to the growthor survival of the pathogen, pest, or insect in an untreated control.Any of the terms of inhibit, destroy, prevent, control, decrease, slow,interfere, resist, or reduce may be used interchangeably. In anembodiment, to “inhibit” is to destroy, prevent, control, reduce,resist, decrease, slow or otherwise interfere with the growth,emergence, or survival of a pathogen, pest, or insect by at least about3% to at least about 100%, or any value in between for example at leastabout 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, 99%, or 100% when compared to the growth orsurvival of the pathogen, pest, or insect in an untreated control. Theamount of inhibition can be measured as described herein or by othermethods known in the art. As used herein, “protects a plant, plant partor seed from a pathogen, pest, or insect pest” is intended to mean thelimiting or eliminating of pathogen, pest, or insect related damage to aplant, plant part or seed by, for example, inhibiting the ability of thepathogen, pest, or insect to grow, emerge, feed, and/or reproduce or bykilling the pathogen, pest, or insect. As used herein, pesticidal and/orinsecticidal activity refers to an activity of a compound, acomposition, and/or a method that protects a plant, a plant part or aseed from a pathogen, pest, or insect.

In an embodiment of the invention, inhibition of a pathogen, pest, orinsect lasts for or provides protection for greater than a day, twodays, three days, four days, five days, six days, a week, two weeks,three weeks, a month or more after an entomopathogenic fungal strain ora composition disclosed herein is applied to subject material. Inanother embodiment, inhibition a pathogen, pest or insect lasts from oneto seven days, from seven to 14 days, from 14 to 21 days, or from 21 to30 days or more. In another embodiment, the inhibition of the growth ofa pathogen, pest, or insect lasts for or provides protection for greaterthan the time from application to adult emergence of the pathogen, pest,or insect.

As used herein, the term “genetically modified” or “geneticallyengineered” is intended to mean any species containing a genetic trait,loci, or sequence that was not found in the species or strain prior tomanipulation. A genetically modified plant may be transgenic, cis-genic,genome edited, or bred to contain a genetic trait, loci, or sequence. Agenetically modified plant may be prepared by means known to thoseskilled in the art, such as transformation by bombardment and/orbacterial mediation, by a Cas/CRISPR or TALENS system, or by breedingtechniques. As used herein, a “trait” is a new or modified locus orsequence of a genetically modified plant, including but not limited to atransgenic plant. A trait may provide herbicide, pathogen, pest, orinsect resistance or any other agronomically desirable characteristic,such as drought resistance or increased nitrogen utilization to thegenetically modified plant.

As used herein, the term “environment of a plant, plant part or a seed”is intended to mean the area surrounding the plant, plant part or seed,including, but not limited to, the soil, the air, or in-furrow. Theenvironment of a plant, plant part or seed may be in close proximity,touching, adjacent to, or in the same field as the plant, plant part orseed. A composition described herein may be applied to the environmentof the plant, plant part or seed as a seed treatment, as a foliarapplication, as an in-furrow application, as a granular application, asa soil application, or as an encapsulated application. As used herein,“in-furrow” is intended to mean within or near the area where a seed isplanted. A composition disclosed herein may be applied in-furrowconcurrently or simultaneously with a seed. In another embodiment, acomposition disclosed herein may be applied sequentially, either beforeor after a seed is planted.

As used herein, the term “different mode of action” is used to refer toa pesticidal composition controlling a pathogen, a pest, or an insectthrough a pathway or receptor that is different from another pesticidalcomposition. As used herein, the term “different mode of action”includes the pesticidal effects of one or more pesticidal compositionsto different binding sites (i.e., different toxin receptors and/ordifferent sites on the same toxin receptor) in the gut membranes ofinsects or through the RNA interference pathway to different targetgenes.

As used herein, the term “pathogen, pest, or insect” includes, but isnot limited to, pathogenic fungi, bacteria, mites, ticks, pathogenicmicroorganisms, and nematodes, as well as insect from the ordersColeoptera, Lepidoptera, Mallophaga, Homoptera, Hemiptera, Orthroptera,Thysanoptera. Dermaptera, Isoptera, Anoplura, Siphonatpera, Trichoptera,and others, including but not limited to Diabrotica virgifera virgifera,Diabrotica undecimpunctata howardi, and Diabrotica barberi.

Embodiments of the present invention are useful in the inhibition oflarvae and adults of the order Coleoptera from the families Anthribidae,Bruchidae and Curculionidae (including, but not limited to: Anthonomusgrandis Boheman (boll weevil); Lissorhoptrus oryzophilus Kuschel (ricewater weevil); Sitophilus granarius Linnaeus (granary weevil); S. oryzaeLinnaeus (rice weevil); Hypera punctata Fabricius (clover leaf weevil);Cylindrocopturus adspersus LeConte (sunflower stem weevil); Smicronyxfulvus LeConte (red sunflower seed weevil); S. sordidus LeConte (graysunflower seed weevil); Sphenophorus maidis Chittenden (maize billbug);flea beetles, cucumber beetles, rootworms, leaf beetles, potato beetlesand leafminers in the family Chrysomelidae (including, but not limitedto: Leptinotarsa decemlineata Say (Colorado potato beetle); Diabroticavirgifera virgifera LeConte (western corn rootworm); D. barberi Smithand Lawrence (northern corn rootworm); D. undecimpunctata howardi Barber(southern corn rootworm); Chaetocnema pulicaria Melsheimer (corn fleabeetle); Phyllotreta cruciferae Goeze (Crucifer flea beetle);Phyllotreta striolata (stripped flea beetle); Colaspis brunnea Fabricius(grape colaspis); Oulema melanopus Linnaeus (cereal leaf beetle);Zygogramma exclamationis Fabricius (sunflower beetle); beetles from thefamily Coccinellidae (including, but not limited to: Epilachnavarivestis Mulsant (Mexican bean beetle); chafers and other beetles fromthe family Scarabaeidae (including, but not limited to: Popilliajaponica Newman (Japanese beetle); Cyclocephala borealis Arrow (northernmasked chafer, white grub); C. immaculata Olivier (southern maskedchafer, white grub); Rhizotrogus majalis Razoumowsky (European chafer);Phyllophaga crinita Burmeister (white grub); Ligyrus gibbosus De Geer(carrot beetle); carpet beetles from the family Dermestidae; wirewormsfrom the family Elateridae, Eleodes spp., Melanotus spp.; Conoderusspp.; Limonius spp.; Agriotes spp.; Ctenicera spp.; Aeolus spp.; barkbeetles from the family Scolytidae and beetles from the familyTenebrionidae.

Methods for measuring pesticidal activity and/or the inhibition of apathogen, pest or insect are well known in the art. See, for example,Czapla and Lang, (1990) J. Econ. Entomol. 83:2480-2485; Andrews, et al.,(1988) Biochem. J. 252:199-206; Marrone, et al., (1985) J. of EconomicEntomology 78:290-293 and U.S. Pat. No. 5,743,477, all of which areherein incorporated by reference in their entirety. Generally, thepesticide is mixed and used in feeding assays. See, for example Marrone,et al., (1985) J. of Economic Entomology 78:290-293. Such assays caninclude contacting plants with one or more pathogens, pests or insectsand determining the plant's ability to survive and/or cause the death ofthe pathogens, pests or insects.

As used herein, the term “plant” refers to all plants, plant parts, andplant populations, such as desirable and undesirable wild plants,cultivars, transgenic plants, and plant varieties (whether or notprotectable by plant variety or plant breeder's rights). Cultivars andplant varieties can be plants obtained by conventional propagation andbreeding methods that can be assisted or supplemented by one or morebiotechnological methods such as by use of double haploids, protoplastfusion, random and directed mutagenesis, molecular or genetic markers orby bioengineering and genetic engineering methods.

The embodiments of the invention may generally be used for any plantspecies, including, but not limited to, monocots and dicots. Examples ofplants of interest include, but are not limited to, corn (Zea mays),Brassica sp. (e.g., B. napus, B. rapa, B. juncea), particularly thoseBrassica species useful as sources of seed oil, alfalfa (Medicagosativa), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghumbicolor, Sorghum vulgare), millet (e.g., pearl millet (Pennisetumglaucum), proso millet (Panicum miliaceum), foxtail millet (Setariaitalica), finger millet (Eleusine coracana)), sunflower (Helianthusannuus), safflower (Carthamus tinctorius), wheat (Triticum aestivum),soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanumtuberosum), peanuts (Arachis hypogaea), cotton (Gossypium barbadense,Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava (Manihotesculenta), coffee (Coffea spp.), coconut (Cocos nucifera), pineapple(Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobroma cacao),tea (Camellia sinensis), banana (Musa spp.), avocado (Persea americana),fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica),olive (Olea europaea), papaya (Carica papaya), cashew (Anacardiumoccidentale), macadamia (Macadamia integrifolia), almond (Prunusamygdalus), sugar beets (Beta vulgaris), sugarcane (Saccharum spp.),oats, barley, vegetables ornamentals, and conifers.

As used herein, the term “plant parts” refers to all above ground andbelow ground parts and organs of plants such as shoot, leaf, blossom androot, for example leaves, needles, stems, branches, blossoms, fruitingbodies, fruits and seeds, as well as roots, tubers, corms and rhizomesare included. Crops and vegetative and generative propagating material,for example, cuttings, corms, rhizomes, tubers, runners and seeds arealso plant parts.

As used herein, the term “spore” includes, but is not limited toconidiospores, aerial conidospores, submerged spores, blastospores,spores, conidia, mycelia, vegetative mycelium, microsclerotia, and/orany other life cycle stage or mixture of life cycle stages of a fungalentomopathogen. An “aerial conidiospore” (AC) refers to conidiosporesformed by the asexual developmental cycle on the surface of an agarmedium, or other solid substrate of appropriate composition. As usedherein, term “submerged spores” refers to submerged conidiospores and/orblastospores that develop in liquid culture.

As used herein, the term “viable” refers to a microbial cell, propagule,or spore that is metabolically active or able to grow and differentiate.Thus, propagules, such as spores, are “viable” when they are dormant andcapable of germinating.

Biological control of pathogens, pests or insects of agriculturalsignificance using a microbial agent, such as fungi, bacteria, oranother species of insect or a biological compound or a biological agentderived from fungi, bacteria, insects and plants affords anenvironmentally friendly and commercially attractive alternative tosynthetic chemical pesticides. Generally speaking, the use ofbiopesticides presents a lower risk of pollution and environmentalhazards and biopesticides provide greater target specificity than ischaracteristic of traditional broad-spectrum chemical insecticides andpesticides. In addition, biopesticides often cost less to produce andthus improve economic yield for a wide variety of crops.

Certain species of microorganisms of the genus Bacillus are known topossess pesticidal activity against a range of insect pests includingLepidoptera, Diptera, Coleoptera, Hemiptera and others. Bacillusthuringiensis (Bt) and Bacillus popilliae are among the most successfulbiocontrol agents discovered to date. Insect pathogenicity has also beenattributed to strains of B. larvae, B. lentimorbus, B. sphaericus and B.cereus. Microbial insecticides, particularly those obtained fromBacillus strains, have played an important role in agriculture asalternatives to chemical pest control.

Crop plants have been developed with enhanced insect resistance bygenetically engineering crop plants to produce pesticidal proteins fromBacillus. For example, corn and cotton plants have been geneticallyengineered to produce pesticidal proteins isolated from strains of Bt.These genetically modified crops are now widely used in agriculture andhave provided the farmer with an environmentally friendly alternative totraditional insect-control methods. While they have proven to be verysuccessful commercially, these genetically modified, insect-resistantcrop plants provide resistance to only a narrow range of theeconomically important insect pests. In some cases, insects can developresistance to different insecticidal compounds, which raises the need toidentify alternative biological control agents for pathogen, pest orinsect control.

An embodiment of the invention relates to an entomopathogenic fungalstrain and/or a composition and a method of making and using theentomopathogenic fungal strain and/or a composition. In an embodimentthe entomopathogenic strains find use in inhibiting, controlling, orkilling a pathogen, pest, or insect, including, but is not limited to,fungi, pathogenic fungi, bacteria, mites, ticks, pathogenicmicroorganisms, and nematodes, as well as insects from the ordersColeoptera, Diptera, Hymenoptera, Lepidoptera, Mallophaga, Homoptera,Hemiptera Orthroptera, Thysanoptera, Dermaptera, Isoptera, Anoplura,Siphonaptera, Trichoptera, etc., particularly Coleoptera, including butnot limited to Diabrotica virgifera virgifera, Diabroticaundecimpunctata howardi, and Diabrotica barberi, and for producingcompositions with pesticidal activity.

In an embodiment, the entomopathogenic fungal strain(s) are selectedfrom the group consisting of: Metarhizium anisopliae 15013-1,Metarhizium robertsii 23013-3, Metarhizium anisopliae 3213-1, andcombinations thereof.

Metarhizium anisopliae 15013-1 (NRRL 67073) was deposited on Jun. 18,2015 at the Agricultural Research Service Culture Collection (NRRL),1815 North University Street, Peoria, Ill., 67073 and given accessionnumber NRRL 67073. The deposits were made under the provisions of theBudapest Treaty on the International Recognition of the Deposit ofMicroorganisms for the Purposes of Patent Procedure.

Metarhizium robertsii 23013-3 (NRRL 67075) was deposited on Jun. 18,2015 at the Agricultural Research Service Culture Collection (NRRL),1815 North University Street, Peoria, Ill., 61604 and given accessionnumber NRRL 67075. The deposits were made under the provisions of theBudapest Treaty on the International Recognition of the Deposit ofMicroorganisms for the Purposes of Patent Procedure.

Metarhizium anisopliae 3213-1 (NRRL 67074) was deposited on Jun. 18,2015 at the Agricultural Research Service Culture Collection (NRRL),1815 North University Street, Peoria, Ill., 61604 and given accessionnumber NRRL 67074. The deposits were made under the provisions of theBudapest Treaty on the International Recognition of the Deposit ofMicroorganisms for the Purposes of Patent Procedure.

An embodiment of the invention relates to a composition comprising orconsisting of or consisting essentially of an entomopathogenic fungalstrain selected from the group consisting of: Metarhizium anisopliae15013-1, Metarhizium robertsii 23013-3, and Metarhizium anisopliae3213-1 and combinations thereof. In another embodiment, a compositioncomprises, consists of, or consists essentially of at least two or moreentomopathogenic fungal strains selected from the group consisting of:Metarhizium anisopliae 15013-1, Metarhizium robertsii 23013-3,Metarhizium anisopliae 3213-1. In a further embodiment, a compositioncomprises, consists of, or consists essentially of an entomopathogenicfungal strain selected from the group consisting of: Metarhiziumanisopliae 15013-1, Metarhizium robertsii 23013-3, and Metarhiziumanisopliae 3213-1 and combinations thereof. In an embodiment, acomposition is a biologically pure culture of Metarhizium anisopliae15013-1, Metarhizium robertsii 23013-3, and Metarhizium anisopliae3213-1 and combinations thereof.

An embodiment of the invention relates to a composition comprising anentomopathogenic fungal strain disclosed herein and a compound or agentselected from the group consisting of: agrochemically active compounds,biocontrol agents, lipo-chitooligosaccharide compounds (LCOs),isoflavones, quinazolines, insecticidal compounds,azolopyrimidinylamines, polymeric compounds, ionic compounds,substituted thiophenes, substituted dithiines, fluopyramm,enaminocarbonyl compounds, strigolactone compounds, anddithiino-tetracarboximide compounds and combinations thereof.

A further embodiment relates to a use of a first composition comprisingan entomopathogenic fungal strain disclosed herein and a secondcomposition comprising a compound or an agent selected from the groupconsisting of: agrochemically active compounds, biocontrol agents,lipo-chitooligosaccharide compounds (LCOs), isoflavones, quinazolines,insecticidal compounds, azolopyrimidinylamine, polymeric compounds,ionic compounds, substituted thiophenes, substituted dithiines,fluopyramm, enaminocarbonyl compounds, strigolactone compounds, anddithiino-tetracarboximide compounds and combinations thereof.

In an embodiment, a composition comprising an entomopathogenic fungalstrain disclosed herein and a biocontrol agent is disclosed. As usedherein, the term “biocontrol agent” (“BCA”) includes bacteria, fungi oryeasts, protozoans, viruses, entomopathogenic nematodes, and botanicalextracts, or products produced by microorganisms including proteins orsecondary metabolites, and inoculants that have one or more of thefollowing characteristics: (1) it inhibits or reduces plant infestationand/or growth of pathogens, pests, or insects, including, but notlimited to, pathogenic fungi, bacteria, and nematodes, as well asarthropod pests such as insects, arachnids, chilopods, diplopods, or itinhibits plant infestation and/or growth of a combination of plantpathogens, pests, or insects; (2) it improves plant performance; (3) itimproves plant yield; (4) it improves plant vigor; and (5) it improvesplant health.

In an embodiment, a composition comprising an entomopathogenic fungalstrain disclosed herein and an agrochemically active compounds isdisclosed. Agrochemically active compounds are substances that are ormay be used for treating a seed, a plant, a plant part, or theenvironment of a seed or a plant or a plant part including, but notlimited to, fungicides, bactericides, insecticides, acaricides,nematicides, molluscicides, safeners, plant growth regulators, plantnutrients, chemical entities with a known mechanism of action,additional microorganisms, and biocontrol agents and combinationsthereof.

In another embodiment, a first composition comprising anentomopathogenic fungal strain disclosed herein and a second compositioncomprising an agrochemically active compound, wherein the first and thesecond composition may inhibit plant pathogens, pests, or insects and/orimprove plant performance is disclosed.

In an embodiment, a first and a second composition disclosed herein canbe applied at the same time to a seed, a plant, a plant part, or theenvironment of a seed, a plant, or a plant part. In another embodiment,a first composition can be applied to a seed, a plant, a plant part, orthe environment of a seed, a plant, or a plant part followed byapplication of a second composition to a seed, a plant, a plant part, orthe environment of a seed, a plant, or a plant part. In yet anotherembodiment, a second composition can be applied to a seed, a plant, aplant part, or the environment of a seed, a plant, or a plant partfollowed by application of a first composition to a seed, a plant, aplant part, or the environment of a seed, a plant, or a plant part.

In an embodiment, a first and a second composition disclosed herein canbe applied at the same time to a seed. In another embodiment, a firstcomposition can be applied to a seed followed by application of a secondcomposition to the seed. In yet another embodiment, a second compositioncan be applied to a seed followed by application of a first compositionto the seed. In another embodiment, a first composition can be appliedto a seed followed by application of a second composition to a plant. Inyet another embodiment, a second composition can be applied to a seedfollowed by application of a first composition to a plant. In anotherembodiment, a first composition can be applied to a seed followed byapplication of a second composition to a plant part. In yet anotherembodiment, a second composition can be applied to a seed followed byapplication of a first composition to a plant part. In anotherembodiment, a first composition can be applied to a seed followed byapplication of a second composition to the environment of the seed. Inyet another embodiment, a second composition can be applied to a seedfollowed by application of a first composition to the environment of theseed. In another embodiment, a first composition can be applied to aseed followed by application of a second composition to the environmentof a plant. In yet another embodiment, a second composition can beapplied to a seed followed by application of a first composition to theenvironment of a plant. In another embodiment, a first composition canbe applied to a seed followed by application of a second composition tothe environment of a plant part. In yet another embodiment, a secondcomposition can be applied to a seed followed by application of a firstcomposition to the environment of a plant part.

In an embodiment, a first and a second composition disclosed herein canbe applied at the same time to a plant. In another embodiment, a firstcomposition can be applied to a plant followed by application of asecond composition to the plant. In yet another embodiment, a secondcomposition can be applied to a plant followed by application of a firstcomposition to the plant. In another embodiment, a first composition canbe applied to a plant followed by application of a second composition toa seed. In yet another embodiment, a second composition can be appliedto a plant followed by application of a first composition to a seed. Inanother embodiment, a first composition can be applied to a plantfollowed by application of a second composition to a plant part. In yetanother embodiment, a second composition can be applied to a plantfollowed by application of a first composition to a plant part. Inanother embodiment, a first composition can be applied to a plantfollowed by application of a second composition to the environment of aseed. In yet another embodiment, a second composition can be applied toa plant followed by application of a first composition to theenvironment of a seed. In another embodiment, a first composition can beapplied to a plant followed by application of a second composition tothe environment of the plant. In yet another embodiment, a secondcomposition can be applied to a plant followed by application of a firstcomposition to the environment of the plant. In another embodiment, afirst composition can be applied to a plant followed by application of asecond composition to the environment of a plant part. In yet anotherembodiment, a second composition can be applied to a plant followed byapplication of a first composition to the environment of a plant part.

In an embodiment, a first and a second composition disclosed herein canbe applied at the same time to a plant part. In another embodiment, afirst composition can be applied to a plant part followed by applicationof a second composition to the plant part. In yet another embodiment, asecond composition can be applied to a plant part followed byapplication of a first composition to the plant part. In anotherembodiment, a first composition can be applied to a plant part followedby application of a second composition to a seed. In yet anotherembodiment, a second composition can be applied to a plant part followedby application of a first composition to a seed. In another embodiment,a first composition can be applied to a plant part followed byapplication of a second composition to a plant. In yet anotherembodiment, a second composition can be applied to a plant part followedby application of a first composition to a plant. In another embodiment,a first composition can be applied to a plant part followed byapplication of a second composition to the environment of a seed. In yetanother embodiment, a second composition can be applied to a plant partfollowed by application of a first composition to the environment of aseed. In another embodiment, a first composition can be applied to aplant part followed by application of a second composition to theenvironment of a plant. In yet another embodiment, a second compositioncan be applied to a plant part followed by application of a firstcomposition to the environment of a plant. In another embodiment, afirst composition can be applied to a plant part followed by applicationof a second composition to the environment of the plant part. In yetanother embodiment, a second composition can be applied to a plant partfollowed by application of a first composition to the environment of theplant part.

In an embodiment, a first and a second composition disclosed herein canbe applied at the same time to the environment of a seed. In anotherembodiment, a first composition can be applied to the environment of aseed followed by application of a second composition to the environmentof the seed. In yet another embodiment, a second composition can beapplied to the environment of a seed followed by application of a firstcomposition to the environment of the seed. In another embodiment, afirst composition can be applied to the environment of a seed followedby application of a second composition to a seed. In yet anotherembodiment, a second composition can be applied to the environment of aseed followed by application of a first composition to a seed. Inanother embodiment, a first composition can be applied to theenvironment of a seed followed by application of a second composition toa plant. In yet another embodiment, a second composition can be appliedto the environment of a seed followed by application of a firstcomposition to a plant. In another embodiment, a first composition canbe applied to the environment of a seed followed by application of asecond composition to a plant part. In yet another embodiment, a secondcomposition can be applied to the environment of a seed followed byapplication of a first composition to a plant part. In anotherembodiment, a first composition can be applied to the environment of aseed followed by application of a second composition to the environmentof a plant. In yet another embodiment, a second composition can beapplied to the environment of a seed followed by application of a firstcomposition to the environment of a plant. In another embodiment, afirst composition can be applied to the environment of a seed followedby application of a second composition to the environment of a plantpart. In yet another embodiment, a second composition can be applied tothe environment of a seed followed by application of a first compositionto the environment of a plant part.

In an embodiment, a first and a second composition disclosed herein canbe applied at the same time to the environment of a plant. In anotherembodiment, a first composition can be applied to the environment of aplant followed by application of a second composition to the environmentof the plant. In yet another embodiment, a second composition can beapplied to the environment of a plant followed by application of a firstcomposition to the environment of the plant. In another embodiment, afirst composition can be applied to the environment of a plant followedby application of a second composition to a seed. In yet anotherembodiment, a second composition can be applied to the environment of aplant followed by application of a first composition to a seed. Inanother embodiment, a first composition can be applied to theenvironment of a plant followed by application of a second compositionto a plant. In yet another embodiment, a second composition can beapplied to the environment of a plant followed by application of a firstcomposition to a plant. In another embodiment, a first composition canbe applied to the environment of a plant followed by application of asecond composition to a plant part. In yet another embodiment, a secondcomposition can be applied to the environment of a plant followed byapplication of a first composition to a plant part. In anotherembodiment, a first composition can be applied to the environment of aplant followed by application of a second composition to the environmentof a seed. In yet another embodiment, a second composition can beapplied to the environment of a plant followed by application of a firstcomposition to the environment of a seed. In another embodiment, a firstcomposition can be applied to the environment of a plant followed byapplication of a second composition to the environment of a plant part.In yet another embodiment, a second composition can be applied to theenvironment of a plant followed by application of a first composition tothe environment of a plant part.

In an embodiment, a first and a second composition disclosed herein canbe applied at the same time to the environment of a plant part. Inanother embodiment, a first composition can be applied to theenvironment of a plant part followed by application of a secondcomposition to the environment of the plant part. In yet anotherembodiment, a second composition can be applied to the environment of aplant part followed by application of a first composition to theenvironment of the plant part. In another embodiment, a firstcomposition can be applied to the environment of a plant part followedby application of a second composition to a seed. In yet anotherembodiment, a second composition can be applied to the environment of aplant part followed by application of a first composition to a seed. Inanother embodiment, a first composition can be applied to theenvironment of a plant part followed by application of a secondcomposition to a plant. In yet another embodiment, a second compositioncan be applied to the environment of a plant part followed byapplication of a first composition to a plant. In another embodiment, afirst composition can be applied to the environment of a plant partfollowed by application of a second composition to the environment of aseed. In yet another embodiment, a second composition can be applied tothe environment of a plant part followed by application of a firstcomposition the environment of a seed. In another embodiment, a firstcomposition can be applied to the environment of a plant part followedby application of a second composition to the environment of a plant. Inyet another embodiment, a second composition can be applied to theenvironment of a plant part followed by application of a firstcomposition to the environment of a plant.

In an embodiment, the use of an entomopathological fungal straindisclosed herein with a composition comprising an insecticidal proteinfrom Pseudomonas sp. such as PSEEN3174 (Monalysin; (2011) PLoS Pathogens7:1-13); from Pseudomonas protegens strain CHA0 and Pf-5 (previouslyfluorescens) (Pechy-Tarr, (2008) Environmental Microbiology10:2368-2386; GenBank Accession No. EU400157); from PseudomonasTaiwanensis (Liu, et al., (2010) J. Agric. Food Chem., 58:12343-12349)and from Pseudomonas pseudoalcligenes (Zhang, et al., (2009) Annals ofMicrobiology 59:45-50 and Li, et al., (2007) Plant Cell Tiss. OrganCult. 89:159-168); insecticidal proteins from Photorhabdus sp. andXenorhabdus sp. (Hinchliffe, et al., (2010) The Open Toxicology Journal,3:101-118 and Morgan, et al., (2001) Applied and Envir. Micro.67:2062-2069); U.S. Pat. Nos. 6,048,838, and 6,379,946; a PIP-1polypeptide of US Patent Publication US20140007292; an AfIP-1A and/orAfIP-1B polypeptide of US Patent Publication US20140033361; a PHI-4polypeptide of U.S. Ser. No. 13/839,702; a PIP-47 polypeptide of PCTSerial Number PCT/US14/51063, a PIP-72 polypeptide of PCT Serial NumberPCT/US14/55128, and S-endotoxins including, but not limited to, theCry1, Cry2, Cry3, Cry4, Cry5, Cry6, Cry7, Cry8, Cry9, Cry10, Cry11,Cry12, Cry13, Cry14, Cry15, Cry16, Cry17, Cry18, Cry19, Cry20, Cry21,Cry22, Cry23, Cry24, Cry25, Cry26, Cry27, Cry 28, Cry 29, Cry 30, Cry31,Cry32, Cry33, Cry34, Cry35, Cry36, Cry37, Cry38, Cry39, Cry40, Cry41,Cry42, Cry43, Cry44, Cry45, Cry 46, Cry47, Cry49, Cry 51 and Cry55classes of 8-endotoxin genes and the B. thuringiensis cytolytic Cyt1 andCyt2 genes is disclosed.

In an embodiment, a composition disclosed herein comprises a silencingconstruct of a polynucleotide of interest resulting in suppression of atarget pathogen, pest, or insect polypeptides. By “silencing element” isit intended to mean a polynucleotide which when contacted by or ingestedby a pest, is capable of reducing or eliminating the level or expressionof a target polynucleotide or the polypeptide encoded thereby. Thesilencing element employed can reduce or eliminate the expression levelof the target sequence by influencing the level of the target RNAtranscript or, alternatively, by influencing translation and therebyaffecting the level of the encoded polypeptide. Silencing elements mayinclude, but are not limited to, a sense suppression element, anantisense suppression element, a double stranded RNA, a siRNA, anamiRNA, a miRNA, or a hairpin suppression element.

In another embodiment, a composition disclosed herein comprises nucleicacid molecules including silencing elements for targeting the vacuolarATPase H subunit, useful for controlling a coleopteran pest populationand infestation as described in US Patent Application Publication2012/0198586. In an embodiment, a composition disclosed herein comprisesribonucleic acid (RNA or double stranded RNA) that inhibits or downregulates the expression of a target gene that encodes: an insectribosomal protein such as the ribosomal protein L19, the ribosomalprotein LAO or the ribosomal protein S27A; an insect proteasome subunitsuch as the Rpn6 protein, the Pros 25, the Rpn2 protein, the proteasomebeta 1 subunit protein or the Pros beta 2 protein; an insect β-coatomerof the COPI vesicle, the γ-coatomer of the COPI vesicle, the β′-coatomerprotein or the ζ-coatomer of the COPI vesicle; an insect Tetraspanine 2A protein which is a putative transmembrane domain protein; an insectprotein belonging to the actin family such as Actin 5C; an insectubiquitin-5E protein; an insect Sec23 protein which is a GTPaseactivator involved in intracellular protein transport; an insectcrinkled protein which is an unconventional myosin which is involved inmotor activity; an insect crooked neck protein which is involved in theregulation of nuclear alternative mRNA splicing; an insect vacuolarH+-ATPase G-subunit protein and an insect Tbp-1 such as Tat-bindingprotein as described in PCT Publication WO 2012/055982. In anotherembodiment, a composition disclosed herein comprises ribonucleic acid(RNA or double stranded RNA) that inhibits or down regulates theexpression of a target gene that encodes Snf7 as described in PCTpublication WO 2007/035650. In an embodiment, a composition disclosedherein comprises polynucleotide silencing elements targeting RPS10described in US Patent Application publication 2011/0054007. In anotherembodiment, a composition disclosed herein comprises polynucleotidesilencing elements targeting RyanR and PAT3 described in US PatentApplication publications 2014/0275208 and US2015/0257389. In anembodiment, a composition disclosed herein comprises interferingribonucleic acids (RNA or double stranded RNA) that functions uponuptake by an insect pest species to down-regulate expression of a targetgene in said insect pest, wherein the RNA comprises at least onesilencing element wherein the silencing element is a region ofdouble-stranded RNA comprising annealed complementary strands, onestrand of which comprises or consists of a sequence of nucleotides whichis at least partially complementary to a target nucleotide sequencewithin the target gene as described in US Patent ApplicationPublications 2012/029750, US 20120297501, and 2012/0322660. In anotherembodiment, a composition disclosed herein comprises potential targetsfor interfering double stranded ribonucleic acids for inhibitinginvertebrate pests including: a Chd3 Homologous Sequence, a Beta-TubulinHomologous Sequence, a 40 kDa V-ATPase Homologous Sequence, a EF1αHomologous Sequence, a 26S Proteosome Subunit p28 Homologous Sequence, aJuvenile Hormone Epoxide Hydrolase Homologous Sequence, a SwellingDependent Chloride Channel Protein Homologous Sequence, aGlucose-6-Phosphate 1-Dehydrogenase Protein Homologous Sequence, anAct42A Protein Homologous Sequence, a ADP-Ribosylation Factor 1Homologous Sequence, a Transcription Factor IIB Protein HomologousSequence, a Chitinase Homologous Sequences, a Ubiquitin ConjugatingEnzyme Homologous Sequence, a Glyceraldehyde-3-Phosphate DehydrogenaseHomologous Sequence, an Ubiquitin B Homologous Sequence, a JuvenileHormone Esterase Homolog, and an Alpha Tubulin Homologous Sequence asdescribed in US Patent Application Publication 2012/0164205.

An embodiment of the invention comprises an additional component, whichmay be a carrier, an adjuvant, a solubilizing agent, a suspending agent,a diluent, a stabilizer, an oxygen scavenger, an antioxidant, a foodmaterial, an anti-contaminant agent, or combinations thereof.

In another embodiment, an additional component(s) may be required for anapplication to which an entomopathogenic fungal strain or a compositiondisclosed herein is to be utilized. For example, if a strain or acomposition is to be utilized on, or in, an agricultural product, theadditional component(s) may be an agriculturally acceptable carrier, anexcipient, or a diluent and combinations thereof. Likewise, if a strainor a composition is to be utilized on, or in, a foodstuff the additionalcomponent(s) may be an edible carrier, excipient or diluent andcombinations thereof.

“Carriers” or “vehicles” mean materials suitable for compoundadministration and include any such materials known in the art such as,for example, any liquid, gel, solvent, liquid diluent, solubilizer, orthe like, which is non-toxic and does not interact with any componentsof a composition in a deleterious manner.

Examples of nutritionally acceptable or edible carriers include, forexample, water, salt solutions, alcohol, silicone, waxes, petroleumjelly, vegetable oils, polyethylene glycols, propylene glycol,liposomes, sugars, gelatin, lactose, amylose, magnesium stearate, talc,surfactants, silicic acid, viscous paraffin, perfume oil, fatty acidmonoglycerides and diglycerides, petroethral fatty acid esters,hydroxymethyl-cellulose, polyvinylpyrrolidone, and the like andcombinations thereof.

Examples of excipients include, but are not limited to, microcrystallinecellulose and other celluloses, lactose, sodium citrate, calciumcarbonate, dibasic calcium phosphate, glycine, starch, milk sugar, andhigh molecular weight polyethylene glycols and combinations thereof.

Examples of diluents include, but are not limited to, water, ethanol,propylene glycol and glycerin, and combinations thereof.

Additional components may be used simultaneously with anentomopathogenic fungal strain and/or a composition disclosed herein(e.g. when they are in admixture together or even when they aredelivered by different routes) or sequentially (e.g. they may bedelivered by different routes).

An entomopathogenic fungal strain and/or a composition disclosed hereinand/or its diluent may also contain chelating agents such as EDTA,citric acid, tartaric acid, etc. Moreover, an entomopathogenic fungalstrain and/or a composition disclosed herein and/or its diluent maycontain active agents selected from fatty acids esters, such as mono-and diglycerides, non-ionic surfactants, such as polysorbates,phospholipids, etc. An entomopathogenic fungal strain and/or acomposition disclosed herein and/or its diluent may also containemulsifiers, which may enhance the stability of an entomopathogenicfungal strain and/or a composition, especially after dilution.

An entomopathogenic fungal strain and/or a composition disclosed hereinmay be used in any suitable form, whether when used alone or whenpresent in a composition. An entomopathogenic fungal strain and/or acomposition disclosed herein may be formulated in any suitable way toensure that the composition comprises an active entomopathogenic fungalstrain.

An entomopathogenic fungal strain and/or a composition hereof may be inthe form of a dry powder that can be sprinkled on or mixed in with aproduct. Entomopathogenic fungal strains and/or compositions of theembodiments of the invention disclosed herein in the form of a drypowder may include an additive such as microcrystalline cellulose, gumtragacanth, gelatin, starch, lactose, alginic acid, Primojel®, or cornstarch (which can be used as a disintegrating agent).

In yet another embodiment, an entomopathogenic fungal strain and/or acomposition disclosed herein can be a spray-dried fermentatere-suspended in H₂O to a percentage selected from the following: 0.05-1,1-3, 3-5, 5-7, 7-10, 10-15, 15-20, and greater than 20%. In anotherembodiment, a clarification step(s) can be performed prior tospray-drying.

In an embodiment, compositions disclosed herein can comprise asuspension of propagules, such as spores, from an entomopathogenicfungal strain disclosed herein. In an embodiment, a suspension ofpropagules, such as spores, can be in a range of 1×10³ to 1×10¹³ CFU/ml.

In an embodiment, compositions disclosed herein can compriseconcentrated, dried propagules, such as spores, from an entomopathogenicfungal strain disclosed herein. In an embodiment, concentrated, driedspores can be in a range of 1×10³ to 1×10¹⁰ CFU/g.

In an embodiment, an entomopathogenic fungal strain and/or a compositiondisclosed herein can be applied in a wet or a partially or a completelydesiccated form or in a slurry, a gel, or another form.

In at least some embodiments, an entomopathogenic fungal strain and/or acomposition disclosed herein can be freeze-dried or lypholized. In atleast some embodiments, an entomopathogenic fungal strain and/or acomposition disclosed herein can be mixed with a carrier. A carrierincludes, but is not limited to, whey, maltodextrin, sucrose, dextrose,limestone (calcium carbonate), rice hulls, yeast culture, dried starch,clay, and sodium silico aluminate. However, it is not necessary tofreeze-dry an entomopathogenic fungal strain and/or a compositiondisclosed herein before using it. An entomopathogenic fungal strainand/or a composition disclosed herein can also be used with or withoutpreservatives and in a concentrated, an un-concentrated, or a dilutedform. In an embodiment, an entomopathogenic fungal strain and/or acomposition disclosed herein can be in the form of a pellet or abiologically pure pellet.

An entomopathogenic fungal strain and/or a composition disclosed hereincan be added to a carrier. Where used, the carrier(s) and anentomopathogenic fungal strain and/or a composition disclosed herein canbe added to a ribbon or paddle mixer and mixed for about 15 minutes,although the timing can be increased or decreased. The components areblended such that a uniform mixture of an entomopathogenic fungal strainand/or a composition disclosed herein and carrier(s) is produced. Thefinal product is preferably a dry, flowable powder.

In an embodiment, an entomopathogenic fungal strain and/or a compositionmay be formulated as a liquid, a dry powder, or a granule. A dry powderor granules may be prepared by means known to those skilled in the art,such as, in a top-spray fluid bed coater, in a bottom spray Wurster, orby a drum granulation (e.g. high sheer granulation), an extrusion, a pancoating or in a micro-ingredients mixer.

In another embodiment, an entomopathogenic fungal strain and/or acomposition may be provided as a spray-dried or a freeze-dried powder.

In yet another embodiment, an entomopathogenic fungal strain and/or acomposition is in a liquid formulation. Such liquid formulation maycontain one or more of the following: a buffer, a salt, sorbitol, and/orglycerol.

In an embodiment, an entomopathogenic fungal strain and/or a compositiondisclosed herein may be formulated with a physiologically acceptablecarrier selected from maltodextrin, calcined (illite) clay, limestone(calcium carbonate), cyclodextrin, wheat or a wheat component, sucrose,starch, Na₂SO₄, talc, PVA, sorbitol, benzoate, sorbiate, glycerol,sucrose, propylene glycol, 1,3-propane diol, glucose, parabens, sodiumchloride, citrate, acetate, phosphate, calcium, metabisulfite, formateand mixtures thereof.

In an embodiment, an entomopathogenic fungal strain and/or a compositiondisclosed herein may be formulated by encapsulation technology toimprove fungal propagule, such as spores, stability and as a way toprotect the fungal propagules from seed applied fungicides. In anembodiment the encapsulation technology may comprise a bead polymer fortimed release of fungal propagules, such as spores, over time. In anembodiment, an encapsulated entomopathogenic fungal strain and/or acomposition may be applied in a separate application of beads in-furrowto the seeds. In another embodiment, an encapsulated entomopathogenicfungal strain and/or a composition may be co-applied simultaneously withseeds.

A coating agent usable for the sustained release of microparticles of anencapsulation embodiment of an entomopathogenic fungal strain and/or acomposition disclosed herein may be a substance which is useful forcoating a microgranular form with the substance to be supported thereon.Any coating agent which can form a coating difficulty permeable for thesupported substance may be used in general, without any particularlimitation. For example, higher saturated fatty acid, wax, thermoplasticresin, thermosetting resin and the like may be used.

Examples of useful higher saturated fatty acids include stearic acid,zinc stearate, stearic acid amide and ethylenebis-stearic acid amide.Examples of waxes include synthetic waxes such as polyethylene wax,carbon wax, Hoechst wax, and fatty acid ester; natural waxes such ascarnauba wax, bees wax and Japan wax; and petroleum waxes such asparaffin wax and petrolatum. Examples of thermoplastic resins includeacrylic ester resin, polyolefins such as polyethylene, polypropylene,polybutene and polystyrene; vinyl polymers such as polyvinyl acetate,polyvinyl chloride, polyvinylidene chloride, polyacrylic acid,polymethacrylic acid, polyacrylate and polymethacrylate; diene polymerssuch as butadiene polymer, isoprene polymer, chloroprene polymer,butadiene-styrene copolymer, ethylene-propylene-diene copolymer,styrene-isoprene copolymer, MMA-butadiene copolymer andacrylonitrile-butadiene copolymer; polyolefin copolymers such asethylene-propylene copolymer, butene-ethylene copolymer,butene-propylene copolymer, ethylene-vinyl acetate copolymer,ethylene-acrylic acid copolymer, styreneacrylic acid copolymer,ethylene-methacrylic acid copolymer, ethylene-methacrylic estercopolymer, ethylene-carbon monoxide copolymer, ethylene-vinylacetate-carbon monoxide copolymer, ethylene-vinyl acetate-vinyl chloridecopolymer and ethylene-vinyl acetate-acrylic copolymer; and vinylchloride copolymers such as vinyl chloride-vinyl acetate copolymer andvinylidene chloride-vinyl chloride copolymer. Examples of thermosettingresins include polyurethane resin, epoxy resin, alkyd resin, unsaturatedpolyester resin, phenolic resin, urea-melamine resin, urea resin andsilicone resin. Of these resins, thermoplastic acrylic ester resin,butadiene-styrene copolymer resin, thermosetting polyurethane resin andepoxy resin are preferred, and among these preferred resins,thermosetting polyurethane resin is particularly preferred. Thesecoating agents can be used either singly or in combinations.

In an embodiment, an entomopathogenic fungal strain and/or a compositioncan include a seed, a part of a seed, a plant, or a plant part.

All plants, plant parts, seeds, seed parts or soil can be treated withan entomopathogenic fungal strain and/or a composition and by a methoddisclosed herein. A composition disclosed herein can include a plant, aplant part, a seed, a seed part, soil, or the environment of a plant, aplant part, a seed, or a seed part. An entomopathogenic fungal strain, acomposition, and a method disclosed herein can be applied to a seed, aseed part, a plant, a plant part, a fruit, the environment of a seed, aseed part, a plant, a plant part, or a fruit, or the soil in which aseed, a seed part, a plant, a plant part or a fruit grows.

An embodiment of the invention relates to a method for reducing plantpathogen, pest, or insect damage to a seed, a plant or a plant partcomprising: (a) treating a seed with an entomopathogenic fungal strainand/or a composition disclosed herein prior to planting. In anotherembodiment, the method further comprises: (b) treating a plant, a plantpart, or the environment of a plant or a plant part obtained from theseed with an entomopathogenic fungal strain and/or a compositiondisclosed herein. The entomopathogenic fungal strain and/or thecomposition used in step (a) may be the same as or different from theentomopathogenic fungal strain or the composition used in step (b).

An embodiment of the invention relates to a method for reducing plantpathogen, pest, or insect damage to a seed, a plant or a plant partcomprising: (a) treating the soil surrounding a seed, a plant or a plantpart with an entomopathogenic fungal strain or a composition disclosedherein. In another embodiment, the method further comprises: (b)treating a seed, a plant a plant part, or the environment of a seed, aplant or a plant panr with an entomopathogenic fungal strain or acomposition disclosed herein. The entomopathogenic fungal strain or thecomposition used in step (a) may be the same as or different from theentomopathogenic fungal strain or the composition used in step (b).

An embodiment of the invention relates to a method for reducing plantpathogen, pest, or insect damage to a seed, a plant or a plant partcomprising: (a) treating a seed prior to planting with anentomopathogenic fungal strain or a composition disclosed herein. Inanother embodiment, the method further comprises: (b) treating the soilsurrounding a seed, a plant or a plant part or the environment of aplant or a plant part with an entomopathogenic fungal strain or acomposition disclosed herein. In still another embodiment, the methodfurther comprises: (c) treating a plant, a plant part, or theenvironment of a plant or a plant part of a plant produced from the seedwith an entomopathogenic fungal strain or a composition disclosedherein. The entomopathogenic fungal strain or the composition used instep (a) may be the same as or different from the entomopathogenicfungal strain or the composition used in step (b). The entomopathogenicfungal strain or the composition used in step (a) may be the same as ordifferent from the entomopathogenic fungal strain or the compositionused in step (c). The entomopathogenic fungal strain or the compositionused in step (b) may be the same as or different from theentomopathogenic fungal strain or the composition used in step (c).

In an embodiment, wild plant species and plant cultivars, or thoseobtained by conventional biological breeding, such as crossing orprotoplast fusion, seeds and plant parts thereof, can be treated with anentomopathogenic fungal strain and/or a composition and a methoddisclosed herein. In another embodiment, transgenic plants and plantcultivars obtained by genetic engineering, seeds and plant partsthereof, can be treated with an entomopathogenic fungal strain and/or acomposition and a method disclosed herein.

In another embodiment, a seed, a plant, a plant part or a plant cultivar(obtained by plant biotechnology methods such as genetic engineering)that may be treated with an entomopathogenic fungal strain and/or acomposition and a method disclosed herein are herbicide-tolerant orherbicide-resistant plants, i.e. plants made tolerant or resistant toone or more herbicides. Such plants can be obtained either by geneticmodification, or by selection of plants containing a mutation impartingsuch herbicide tolerance or resistance. Herbicide-resistant plants arefor example glyphosate-tolerant plants, i.e. plants made tolerant to theherbicide glyphosate or salts thereof. Plants can be made tolerant toglyphosate through different means. For example, glyphosate-tolerantplants can be obtained by transforming the plant with a gene encodingthe enzyme 5-enolpyruvylshilcimate-3-phosphate synthase (EPSPS).

Seeds, plants, plant parts or plant cultivars (obtained by plantbiotechnology methods such as genetic engineering) that may also betreated with an entomopathogenic fungal strain and/or a composition anda method disclosed herein are insect-resistant genetically modifiedplants (or transgenic plants), i.e. plants made resistant to attack bycertain target insects. Such seeds, plants, plant parts or plantcultivars can be obtained by genetic transformation, or by selection ofplants containing a mutation imparting such insect resistance.

In another embodiment, seeds, plants, plant parts, or plant cultivars(obtained by plant biotechnology methods such as genetic engineering)that may be treated with an entomopathogenic fungal strain and/or acomposition and a method disclosed herein are tolerant to abioticstresses. Such seeds, plants, plant parts, or plant cultivars can beobtained by genetic transformation, or by selection of seeds, plants,plant parts, or plant cultivars containing a mutation imparting suchstress resistance.

In another embodiment, seeds, plants, plant parts, or plant cultivars(obtained by plant biotechnology methods such as genetic engineering)that may be treated with an entomopathogenic fungal strain and/or acomposition and a method disclosed herein are conventionally bred,produced by mutagenesis, or genetically engineered to contain acombination or stack of valuable traits, including, but not limited to,herbicide tolerance, insect resistance, and abiotic stress tolerance.The entomopathogenic fungal strains, compositions and methods of theembodiments of the invention may be used to treat plant varieties whichwill be developed, or marketed, in the future and which have thesegenetic traits or traits to be developed in the future.

As used herein, applying an entomopathogenic fungal strain or acomposition to a seed, a plant, or plant part, or the environment of aseed, a plant or a plant part includes contacting the seed, the plant,or the plant part, or the environment of the seed, the plant or theplant part directly and/or indirectly with the entomopathogenic fungalstrains or the compositions disclosed herein. In an embodiment, anentomopathogenic fungal strain or a composition may be directly appliedto a seed, a plant or a plant part, or the environment of a seed, aplant or a plant part as a spray, a rinse, or a powder, or anycombination thereof. A contacting step may occur while a seed, a plantor a plant part is being grown, while a plant or a plant part is beingfertilized, while a plant or a plant part is being harvested, after aplant or a plant part has been harvested, while a plant or a plant partis being processed, while a plant or a plant part is being packaged, orwhile a plant or a plant part is being stored in warehouse or on a shelfin a store.

As used herein, a spray refers to a mist of liquid particles thatcontains an entomopathogenic fungal strain or a composition of anembodiment disclosed herein. In an embodiment, a spray containing anentomopathogenic fungal strain or a composition of an embodimentdisclosed herein may be applied to a seed prior to or at the time ofplanting. In an embodiment, a spray may be applied to a plant or a plantpart while the plant or the plant part is being grown. In anotherembodiment, a spray may be applied to a plant or a plant part while theplant or the plant part is being fertilized. In another embodiment, aspray may be applied to a plant or a plant part while the plant or theplant part is being harvested. In another embodiment, a spray may beapplied to a plant or a plant part after the plant or the plant part hasbeen harvested. In another embodiment, a spray may be applied to a plantor a plant part while the plant or the plant part is being processed. Inanother embodiment, a spray may be applied to a plant or a plant partwhile the plant or the plant part is being packaged. In anotherembodiment, a spray may be applied to a plant or a plant part while theplant or the plant part is being stored.

In another embodiment, an entomopathogenic fungal strain or acomposition disclosed herein may be applied directly to a seed, a plantor a plant part, or the environment of a seed, a plant or a plant partas a rinse. As used herein, a rinse is a liquid containing anentomopathogenic fungal strain or a composition disclosed herein. Such arinse may be poured over a seed, a plant or a plant part or theenvironment of a seed, a plant or a plant part. A seed, a plant or aplant part may also be immersed or submerged in the rinse, then removedand allowed to dry.

In another embodiment, an entomopathogenic fungal strain or acomposition disclosed herein may be applied to a seed, a plant or aplant part or the environment of a seed, a plant or a plant part and maycover 50% of the surface area of the seed, the plant or the plant partor the environment of a seed, a plant or a plant part. In anotherembodiment, an entomopathogenic fungal strain or a composition may beapplied to a seed, a plant or a plant part or the environment of a seed,a plant or a plant part and may cover a percentage of the surface areaof the seed, the plant or the plant part or the environment of a seed, aplant or a plant part selected from the group consisting of: from 50% toabout 95%, from 60% to about 95%, from 70% to about 95%, from 80% toabout 95%, and from 90% to about 95%.

In another embodiment, an entomopathogenic fungal strain or acomposition disclosed herein may cover from about 20% to about 30%, fromabout 30% to about 40%, from about 40% to about 50%, from about 50% toabout 60%, from about 60% to about 70%, from about 70% to about 80%,from about 80% to about 90%, from about 90% to about 95%, from about 95%to about 98%, from about 98% to about 99% or 100% of the surface area ofa seed, a plant or a plant part or the environment of a seed, a plant ora plant part.

In another embodiment, an entomopathogenic fungal strain or acomposition disclosed herein may be applied directly to a seed, a plantor a plant part or the environment of a seed, a plant or a plant part asa powder. As used herein, a powder is a dry or nearly dry bulk solidcomposed of a large number of very fine particles that may flow freelywhen shaken or tilted. A dry or a nearly dry powder compositiondisclosed herein preferably contains a low percentage of water, such as,for example, less than 5%, less than 2.5%, or less than 1% by weight.

In another embodiment, an entomopathogenic fungal strain and/or acomposition disclosed herein can be applied indirectly to a seed, aplant or a plant part or the environment of a seed, a plant or a plantpart. For example, a seed, a plant or a plant part or the environment ofa seed, a plant or a plant part having an entomopathogenic fungal strainand/or a composition disclosed herein already applied may be touchinganother seed, another plant or another plant part so that anentomopathogenic fungal strain or a composition rubs off on the otherseed, plant or plant part. In a further embodiment, an entomopathogenicfungal strain or a composition disclosed herein may be applied using anapplicator. In some embodiments, an applicator may include, but is notlimited to, a syringe, a sponge, a paper towel, or a cloth, or anycombination thereof.

In another embodiment, an entomopathogenic fungal strain and/or acomposition disclosed herein may be a colloidal dispersion. A colloidaldispersion is a type of chemical mixture where one substance isdispersed evenly throughout another. Particles of the dispersedsubstance are only suspended in the mixture, unlike a solution, wherethey are completely dissolved within a solvent. This occurs because theparticles in a colloidal dispersion are larger than the particles in asolution, they are small enough to be dispersed evenly and to maintain ahomogenous appearance, but are large enough to scatter light and notdissolve. Colloidal dispersions are an intermediate between homogeneousand heterogeneous mixtures and are sometimes classified as either“homogeneous” or “heterogeneous” based upon their appearance.

In an embodiment, an entomopathogenic fungal strain, a composition, anda method disclosed herein are suitable for use with a seed. In anotherembodiment, an entomopathogenic fungal strain, a composition, and amethod disclosed herein are suitable for use with a seed of one or moreof any of the plants or the plant parts recited previously.

In still another embodiment, an entomopathogenic fungal strain, acomposition and a method disclosed herein can be used to treattransgenic or genetically modified seed. A transgenic seed refers to theseed of plants containing at least one heterologous gene that allows theexpression of a polypeptide or protein not naturally found in the plant.The heterologous gene in transgenic seed can originate, for example,from microorganisms of the species Bacillus, Rhizobium, Pseudomonas,Serratia, Trichoderma, Clavibacter, Glomus or Gliocladium.

In an embodiment, a seed is treated in a state in which it issufficiently stable so that the treatment does not cause any damage. Ingeneral, treatment of a seed may take place at any point in time betweenharvesting and sowing. In an embodiment, a seed to be treated with anentomopathogenic fugal strain, a composition or a method disclosedherein is separated from a plant and freed from cobs, shells, stalks,coats, hairs or flesh of a fruit. Thus, it is possible to use, forexample, a seed which has been harvested, cleaned and dried.Alternatively, it is also possible to use a seed which, after drying,has been treated, for example, with water and then dried again.

In an embodiment, a seed is treated with an entomopathogenic fungalstrain, a composition and a method disclosed herein in such a way thatthe germination of the seed is not adversely affected and/or a resultingplant is not damaged.

In an embodiment, an entomopathogenic fungal strain, a composition, anda method disclosed herein can be applied directly to a seed. Forexample, an entomopathogenic fungal strain, a composition and a methoddisclosed herein can be applied without additional components andwithout having been diluted.

In another embodiment, an entomopathogenic fungal strain, and/or acomposition disclosed herein are applied to a seed in the form of asuitable formulation. Suitable formulations and methods for thetreatment of a seed are known to the person skilled in the art and aredescribed, for example, in the following documents: U.S. Pat. Nos.4,272,417 A, 4,245,432 A, 4,808,430 A, 5,876,739 A, US 2003/0176428 A1.WO 2002/080675 A1. WO 2002/028186 A2.

An entomopathogenic fungal strain and/or a composition disclosed hereincan be converted into customary seed dressing formulations, such assolutions, emulsions, suspensions, powders, foams, slurries, colloidaldispersions or other coating materials for seed, and also ULVformulations. These formulations are prepared in a known manner bymixing an entomopathogenic fungal strain and/or a composition disclosedherein with customary additives, such as, for example, customaryextenders and also solvents or diluents, colorants, wetting agents,dispersants, emulsifiers, defoamers, preservatives, secondarythickeners, adhesives, gibberellins and water as well.

In another embodiment, suitable colorants may be present in a seeddressing formulation including all colorants customary for suchpurposes. Use may be made both of pigments, which have sparingsolubility in water, and of dyes, which are soluble in water. Examplesinclude the colorants known under the designations Rhodamine B, C.I.Pigment Red 112, and C.I. Solvent Red 1.

In another embodiment, suitable wetting agents may be present in a seeddressing formulation, including all substances that promote wetting in aformulation of active agrochemical substances. The use ofalkylnaphthalene-sulphonates, such as diisopropyl- ordiisobutylnaphthalene-sulphonates are preferred.

In still another embodiment, suitable dispersants and/or emulsifiers maybe present in a seed dressing formulation including all nonionic,anionic, and cationic dispersants. In an embodiment, nonionic or anionicdispersants or mixtures of nonionic or anionic dispersants can be used.In an embodiment, nonionic dispersants include, but are not limited to,ethylene oxide-propylene oxide block polymers, alkylphenol polyglycolethers, and tristyrylphenol polyglycol ethers, and their phosphated orsulphated derivatives.

In still another embodiment, defoamers may be present in a seed dressingformulation incorporating an entomopathogenic fungal strain and/or acomposition of an embodiment of the invention including allfoam-inhibiting compounds including, but not limited to, siliconedefoamers, magnesium stearate, silicone emulsions, long-chain alcohols,fatty acids and their salts, and organofluorine compounds and mixturesthereof.

In still another embodiment, secondary thickeners may be present in aseed dressing formulation including all compounds which can be used forsuch purposes in an agrochemical composition, including, but not limitedto, cellulose derivatives, acrylic acid derivatives, polysaccharides,such as xanthan gum or Veegum®, modified clays, phyllosilicates, such asattapulgite and bentonite, and also finely divided silicic acids.

In another embodiment, adhesives may be present in a seed dressingformulation incorporating an entomopathogenic fungal strain and/or acomposition disclosed herein including all customary binders which canbe used in seed dressings. Polyvinylpyrrolidone, polyvinyl acetate,polyvinyl alcohol and tylose are preferred.

In yet another embodiment, a seed dressing formulation incorporating anentomopathogenic fungal strain and/or a composition disclosed herein maybe used directly or after dilution with water to treat a seed of anyseed type. A seed dressing formulation or its dilute preparation mayalso be used to dress a seed of transgenic plants. In this context,synergistic effects may arise in interaction of the seed dressingformulation with substances formed by transgenic expression.

Suitable mixing equipment for treating a seed with a seed dressingformulation or preparations prepared from them by adding water includesall mixing equipment that can commonly be used for dressing. Thespecific procedure adopted when dressing comprises introducing a seedinto a mixer, adding a particular desired amount of a seed dressingformulation, either as it is or following dilution with water, andcarrying out mixing until the formulation is uniformly distributed onthe seed. Optionally, a drying operation follows the treating operation.

In an embodiment, an entomopathogenic fungal strain and/or a compositionor a formulation disclosed herein can be added to a plant, a plant part,and/or a seed at a rate of about 1×10³ to 1×10¹³ colony forming units(cfu) per seed, including about 1×10³ cfu/seed, or about 1×10⁴ cfu/seed,1×10⁵ cfu/seed, or about 1×10⁶ cfu/seed, or about 1×10⁷ cfu/seed, orabout 1×10⁸ cfu/seed, or about 1×10⁹ cfu/seed, or about 1×10¹⁰ cfu/seed,or about 1×10¹¹ cfu/seed, or about 1×10¹² cfu/seed, or about 1×10¹³cfu/seed including about 1×10³ to 1×10⁸ cfu/seed about 1×10³ to 1×10⁷cfu/seed, about 1×10³ to 1×10⁵ cfu/seed, about 1×10³ to 1×10⁶ cfu/seed,about 1×10³ to 1×10 cfu/seed, about 1×10³ to 1×10⁹ cfu/seed, about 1×10³to 1×10¹⁰ cfu/seed, about 1×10³ to 1×10¹¹ cfu/seed, about 1×10³ to1×10¹² cfu/seed, about 1×10³ to 1×10¹³ cfu/seed, about 1×10⁴ to 1×10⁸cfu/seed about 1×10⁴ to 1×10⁷ cfu/seed, about 1×10⁴ to 1×10⁵ cfu/seed,about 1×10⁴ to 1×10⁶ cfu/seed, about 1×10⁴ to 1×10⁹ cfu/seed, about1×10⁴ to 1×10¹⁰ cfu/seed, about 1×10¹¹ to 1×10⁹ cfu/seed, about 1×10⁴ to1×10¹² cfu/seed about 1×10⁴ to 1×10¹³ cfu/seed, about 1×10⁵ to 1×10⁷cfu/per seed, about 1×10⁵ to 1×10⁶ cfu/per seed, about 1×10⁵ to 1×10⁸cfu/per seed, about 1×10⁵ to 1×10⁹ cfu/per seed, about 1×10⁵ to 1×10¹⁰cfu/per seed, about 1×10⁵ to 1×10¹¹ cfu/per seed, about 1×10⁵ to 1×10¹²cfu/per seed, about 1×10⁵ to 1×10¹³ cfu/per seed, about 1×10⁶ to 1×10⁸cfu/per seed, about 1×10⁶ to 1×10⁷ cfu/per seed, about 1×10⁶ to 1×10⁹cfu/per seed, about 1×10⁶ to 1×10¹⁰ cfu/per seed, about 1×10⁶ to 1×10¹¹cfu/per seed, about 1×10⁶ to 1×10¹² cfu/per seed, about 1×10⁶ to 1×10¹³cfu/per seed, about 1×10⁷ to 1×10⁸ cfu/per seed, about 1×10⁷ to 1×10⁹cfu/per seed, about 1×10⁷ to 1×10¹⁰ cfu/per seed, about 1×10⁷ to 1×10¹¹cfu/per seed, about 1×10⁷ to 1×10¹² cfu/per seed, about 1×10⁷ to 1×10¹³cfu/per seed, about 1×10⁸ to 1×10⁹ cfu/per seed, about 1×10⁸ to 1×10¹⁰cfu/per seed, about 1×10⁸ to 1×10¹¹ cfu/per seed, about 1×10⁸ to 1×10¹²cfu/per seed, about 1×10⁸ to 1×10¹³ cfu/per seed, about 1×10⁹ to 1×10¹⁰cfu/per seed, about 1×10⁹ to 1×10¹¹ cfu/per seed, about 1×10⁹ to 1×10¹²cfu/per seed, about 1×10⁹ to 1×10¹³ cfu/per seed, about 1×10¹⁰ to 1×10¹¹cfu/per seed, about 1×10¹⁰ to 1×10¹² cfu/per seed, about 1×10¹⁰ to1×10¹³ cfu/per seed, about 1×10¹¹ to 1×10¹² cfu/per seed, about 1×10¹¹to 1×10¹³ cfu/per seed, and about 1×10¹² to 1×10¹³ cfu/per seed. As usedherein, the term “colony forming unit” or “cfu” is a unit containingentomopathogen fungal structures capable of growing and producing acolony in favorable conditions. The cfu count serves as an estimate ofthe number of viable structures or cells in a sample.

In an embodiment, an entomopathogenic fungal strain and/or a compositioncan be formulated as a liquid seed treatment comprising anentomopathogenic fungal strain and/or composition disclosed herein. Theseeds are substantially uniformly coated with one or more layers of anentomopathogenic fungal strain, using conventional methods of mixing,spraying or a combination thereof. Application is done using equipmentthat accurately, safely, and efficiently applies a seed treatmentproduct to a seed. Such equipment uses various types of coatingtechnology such as rotary coaters, drum coaters, fluidized bedtechniques, spouted beds, rotary mists or a combination thereof.

In an embodiment, an application is done via either a spinning“atomizer” disk or a spray nozzle that evenly distributes a seedtreatment onto a seed as it moves through the spray pattern. In yetanother embodiment, a seed is then mixed or tumbled for an additionalperiod of time to achieve additional treatment distribution and drying.A seed can be primed or unprimed before coating with an entomopathogenicfungal strain and/or a composition disclosed herein to increase theuniformity of germination and emergence. In an alternative embodiment, adry powder composition can be metered onto a moving seed.

In still another embodiment, a seed may be coated via a continuous or abatch coating process. In a continuous coating process, continuous flowequipment simultaneously meters both the seed flow and the seedtreatment products. A slide gate, cone and orifice, seed wheel, orweight device (belt or diverter) regulates seed flow. Once the seed flowrate through treating equipment is determined, the flow rate of the seedtreatment products is calibrated to the seed flow rate in order todeliver the desired dose to a seed as it flows through the seed treatingequipment. Additionally, a computer system may monitor the seed input tothe coating machine, thereby maintaining a constant flow of theappropriate amount of a seed.

In a batch coating process, batch treating equipment weighs out aprescribed amount of a seed and places the seed into a closed treatingchamber or bowl where a corresponding amount of seed a treatment is thenapplied. The seed and the seed treatment are then mixed to achieve asubstantially uniform coating on each seed. This batch is then dumpedout of the treating chamber in preparation for the treatment of a nextbatch. With computer control systems, this batch process is automatedenabling it to continuously repeat a batch treating process.

A variety of additives can be added to a seed treatment. Binders can beadded and include those composed preferably of an adhesive polymer thatcan be natural or synthetic without phytotoxic effect on a seed to becoated. A variety of colorants may be employed, including organicchromophores classified as nitroso, nitro, azo, including monoazo,bisazo, and polyazo, diphenylmethane, triarylmethane, xanthene, methane,acridine, thiazole, thiazine, indamine, indophenol, azine, oxazine,anthraquinone, and phthalocyanine and combinations thereof. Otheradditives can be added including trace nutrients such as salts of iron,manganese, boron, copper, cobalt, molybdenum, and zinc and combinationsthereof. A polymer or other dust control agent can be applied to retaina treatment on a seed surface.

Other conventional seed treatment additives include, but are not limitedto, coating agents, wetting agents, buffering agents, andpolysaccharides. An agriculturally acceptable carrier can be added to aseed treatment formulation such as water, solids or dry powders. A drypowder can be derived from a variety of materials such as wood barks,calcium carbonate, gypsum, vermiculite, talc, humus, activated charcoal,and various phosphorous compounds.

In an embodiment, a seed coating can comprise of a filler, which is anorganic or an inorganic, a natural or a synthetic component with whichan entomopathological fungal strain and/or a composition hereof iscombined to facilitate its application onto a seed. In an embodiment, afiller is an inert solid such as clays, natural or synthetic silicates,silica, resins, waxes, solid fertilizers (for example ammonium salts),natural soil minerals, such as kaolins, clays, talc, lime, quartz,attapulgite, montmorillonite, bentonite, or diatomaceous earths, orsynthetic minerals, such as silica, alumina, or silicates, in particularaluminum or magnesium silicates and combinations thereof.

In an embodiment, an entomopathogenic fungal strain and/or a compositiondisclosed herein may be formulated by encapsulation technology toimprove fungal spore stability and as a way to protect the fungal sporesfrom seed applied fungicides. In an embodiment the encapsulationtechnology may comprise a bead polymer for timed release of anentomopathogenic fungal strain and/or a composition disclosed hereinover time. In an embodiment, the encapsulation technology may comprise azeolite material. In an embodiment, an encapsulated entomopathogenicfungal strain and/or a composition disclosed herein may be applied in aseparate application of beads in-furrow to the seeds. In anotherembodiment, an encapsulated entomopathogenic fungal strain and/or acomposition disclosed herein may be co-applied along with seedssimultaneously.

Insect resistance management (IRM) is a term used to describe practicesaimed at reducing the potential for insect pests to become resistant toan insect management tactic. Maintenance of the efficacy of a Bt(Bacillus thuringiensis derived pesticidal protein), another pesticidalprotein, a chemical, or a biological agent is necessary for effectiveinsect control. IRM is of great importance in maintaining effectiveinsect control because of the threat insect resistance poses to thefuture use of pesticidal plant-incorporated protectants and insecticidaltrait technology as a whole. Specific IRM strategies, such as a refugestrategy, mitigate insect resistance to specific insecticidal proteinsproduced in corn, soybean, cotton, and other crops. However, suchstrategies result in portions of crops being left susceptible to one ormore insect pests in order to ensure that non-resistant insects developand become available to mate with any resistant insect pests produced ina protected crop. Accordingly, from a farmer/producer's perspective, itis highly desirable to have as small a refuge as possible and yet stillmanage insect resistance, in order that the greatest yield is obtainedwhile still maintaining the efficacy of the insect pest control methodused, whether the method is a Bt pesticidal protein, a differentpesticidal protein, a chemical, a biological agent, some other compoundor method, or combinations thereof.

An often used IRM strategy is the planting of a refuge (a portion of thetotal acreage using non-Bt/pesticidal trait seed), as it iscommonly-believed that this will delay the development of insectresistance to pesticidal traits by maintaining insect susceptibility.The theoretical basis of the refuge strategy for delaying resistancehinges on the assumption that the frequency and recessiveness of insectresistance is inversely proportional to insect pest susceptibility;resistance will be rare and recessive only when insect pests are verysusceptible to a toxin, and conversely resistance will be more frequentand less recessive when insect pests are not very susceptible.Furthermore, the strategy assumes that resistance to an insecticidaltrait is recessive and is conferred by a single locus with two allelesresulting in three genotypes: susceptible homozygotes (SS),heterozygotes (RS), and resistant homozygotes (RR). It also assumes thatthere will be a low initial resistance allele frequency and that therewill be extensive random mating between resistant and susceptibleadults. Under ideal circumstances, only rare RR individuals will survivea pesticidal toxin produced by a crop. Both SS and RS individuals willbe susceptible to the pesticidal toxin. A structured refuge is anon-Bt/insecticidal trait portion of a grower's field or set of fieldsthat provides for the production of susceptible (SS) insects that mayrandomly mate with rare resistant (RR) insects surviving theinsecticidal trait crop, which may be a Bt trait crop, to producesusceptible RS heterozygotes that will be killed by the Bt/insecticidaltrait crop. An integrated refuge is a certain portion of a grower'sfield or set of fields of randomly planted non-Bt/insecticidal traitplants that provides for the production of susceptible (SS) insects thatmay randomly mate with rare resistant (RR) insects surviving theinsecticidal trait crop to produce susceptible RS heterozygotes thatwill be killed by the pesticidal trait crop. Each refuge strategy willremove resistant (R) alleles from an insect population and delay theevolution of resistance.

Another strategy to reduce the need for a refuge is the pyramiding oftraits with different modes of action against a target pathogen, pest orinsect. For example, Bt toxins that have different modes of actionpyramided in one transgenic plant are able to have reduced refugerequirements due to reduced resistance risk. Different modes of actionin a pyramid combination also extend the durability of each trait, asresistance is slower to develop to each trait.

Currently, the size, placement, and management of a refuge are oftenconsidered critical to the success of refuge strategies to mitigateinsect resistance to a Bt/pesticidal trait produced in corn, cotton,soybean, and other crops. Because of the decrease in yield in refugeplanting areas, some farmers choose to eschew the refuge requirements,and others do not follow the size and/or placement requirements. Theseissues result in either no refuge or a less effective refuge, and acorresponding risk of an increase in the development of resistant pests.

Accordingly, there remains a need for methods for managing pestresistance in a plot of pest resistant crop plants. It would be usefulto provide an improved method for the protection of plants, especiallycorn and soybeans as well as other crop plants, from feeding damage bypests. It would be particularly useful if such a method reduced therequired application rate of conventional chemical pesticides andinsecticides, and limited the number of separate field operationsrequired for crop planting and cultivation. In addition, a method ofdeploying a biocontrol agent that increases the durability of aninsecticidal/pesticidal trait or increases the efficacy of a resistancemanagement strategy would be useful.

An embodiment relates to a method of reducing or preventing thedevelopment of resistance to a plant insecticidal/pesticidal compositionof a pest in a population of pests comprising providing a plantprotection composition, such as a Bt insecticidal/pesticidal protein, atransgenic insecticidal/pesticidal protein, another pesticidal protein,a chemical pesticide, or a pesticidal biological agent, to seed, aplant, a plant part or a planted area or an insecticidal trait andproviding an entomopathogenic fungal strain, and/or a composition,and/or a method described herein to the seed, the plant, the plant partor the planted area or the insecticidal trait. Another embodimentrelates to a method of reducing or preventing the development ofresistance to a plant pesticidal/insecticidal trait comprising providinga composition comprising a plant pesticidal/insecticidal trait and anentomopathogenic fungal strain and/or a composition described herein. Afurther embodiment relates to a method of reducing or preventing thedevelopment of resistance to a plant Coleopteran insecticidal traitcomprising providing a composition comprising a plant Coleopteraninsecticidal trait and an entomopathogenic fungal strain and/or acomposition described herein. Another embodiment relates to a method ofreducing or preventing the development of resistance to a plantDiabrotica virgifera virgifera insecticidal trait comprising providing aplant Diabrotica virgifera virgifera insecticidal trait and anentomopathogenic fungal strain and/or a composition described herein.

A further embodiment relates to a method of increasing the durability ofplant pest compositions comprising providing a plant protectioncomposition to a seed, a plant, a plant part or a planted area, andproviding an entomopathogenic fungal strain, and/or a composition,and/or a method described herein to the seed, the plant, the plant partor the planted area, wherein the entomopathogenic fungal strain, and/orthe composition, and/or the method described herein has a different modeof action than the plant protection composition.

In a still further embodiment, a required refuge may be reduced oreliminated by the presence of an entomopathogenic fungal strain, and/ora composition, and/or a method described herein when applied to anon-refuge plant. In another embodiment, a refuge may include anentomopathogenic fungal strain, and/or a composition, and/or a methoddescribed herein as a spray, a bait, or as a different mode ofapplication.

In an embodiment of the invention, a composition that increasesresistance to a pathogen, a pest, or an insect comprises anentomopathogenic fungal strain disclosed herein and a non-Btinsecticidal trait. In another embodiment, an entomopathogenic fungalstrain is selected from the group consisting of: Metarhizium anisopliae15013-1, Metarhizium robertsii 23013-3, and Metarhizium anisopliae3213-1. In another embodiment, a non-Bt insecticidal trait comprises aplant-derived insecticidal protein, a bacterial/archeal-derivedinsecticidal protein not from a Bt (such as a Pseudomonas insecticidalprotein), an animal-derived insecticidal protein, or a silencingelement. In another embodiment of the invention, a composition thatincreases durability of a non-Bt insecticidal trait comprises anentomopathogenic fungal strain and a non-Bt insecticidal trait. Inanother embodiment, a non-Bt insecticidal trait comprises a PIP-72polypeptide of PCT Serial Number PCT/US14/55128. In another embodiment,a non-Bt insecticidal trait comprises a polynucleotide silencing elementtargeting RyanR (US Patent Application publication 2014/0275208). Inanother embodiment, a non-Bt insecticidal trait comprises apolynucleotide silencing element targeting RyanR (US Patent Applicationpublication 2014/0275208) and a PIP-72 polypeptide of PCT Serial NumberPCT/US14/55128.

In a further embodiment, a composition that increases resistance to apathogen, a pest, or an insect comprises an entomopathogenic fungalstrain disclosed herein and a Bt insecticidal trait. These compositionsmay provide to a seed, a plant or a plant part additive or synergisticresistance to a pathogen, a pest, or an insect. A Bt insecticidal traitmay have activity to Coleopteran plant pests, such as Diabroticavirgifera virgifera. In an embodiment of the invention, a compositioncomprises an entomopathogenic fungal strain disclosed herein and a Btinsecticidal trait, wherein the Bt insecticidal trait comprises a Cry3Btoxin disclosed in U.S. Pat. No. 8,101,826, PCT Publications WO2002/028184, WO 2002/030205, WO 2002/028185, and WO 2000/011185, amCry3B toxin disclosed in U.S. Pat. No. 8,269,069 and PCT Publication WO1999/031248, a mCry3A toxin disclosed in PCT Publication WO 2003/018810and U.S. Pat. No. 8,269,069, or a Cry34/35 toxin disclosed in U.S. Pat.Nos. 7,309,785, 7,524,810, 7,985,893 and 7,939,651 and PCT PublicationWO 2001/014417, and transgenic events containing these Bt insecticidaltoxins and other Coleopteran active Bt insecticidal traits for example,event MON863 disclosed in PCT Publication WO 2004/011601, event MIR604disclosed in PCT Publication WO 2005/103301, event 5307 disclosed inU.S. Pat. No. 9,133,474, event DAS-59122 disclosed in PCT Publication WO2006/039376, event DP-4114 disclosed in PCT Publication WO 2013/147018,event MON 87411 disclosed in PCT Publication WO 2013/169923, and eventMON88017 disclosed in PCT Publication WO 2005/059103 all of which areincorporated herein by reference.

The entomopathogenic fungal strains, compositions, and methods will befurther understood by reference to the following non-limiting Examples.The following Examples are provided for illustrative purposes only. TheExamples are included solely to aid in a more complete understanding ofthe described embodiments of the invention. The Examples do not limitthe scope of the embodiments of the invention described or claimed.

Example 1

Growing Metarhizium anisopliae Strain 15013-1 in Liquid Culture at ShakeFlask Scale

Siliconized 4-baffle shake flasks with 50 mL of soy 10C (25% glucose+75%fructose):1N medium (Table 3) were inoculated with 5×10⁶ AC/mL of M.anisopliae strain 15013-1 in duplicate. The culture was incubated at 300rpm and 28° C. for 7 days, samples were taken at day 3, 5 and 7, andfermentation parameters recorded. At the end of the 7-day fermentation6.825×10⁴ Microsclerotia (MS)/mL±0.849×10⁴ MS/mL, 8.000×10⁷ SubmergedSpores (SS)/mL±2.828×10⁷ SS/mL, and DCW of 43.07 g/kg±2.91 g/kg wereproduced in the culture. In addition, a pH of 7.0±0 and glucoseconcentration of 54 mg/mL±16 mg/mL were recorded in the culture (FIG. 1and Table 4).

TABLE 4 DCW, pH and glucose concentration at end of liquid fermentationof M. anisopliae strain 15013-1 in soy 10 C. (25% glucose + 75%fructose):1N medium after 7 days of incubation at 300 rpm and 28° C., atshake flask scale. M. anisopliae strain 15013-1 Average StDev DCW [g/kg]43.07366 2.90557 pH 7 0 Glucose [mg/L] 54.00000 15.55635

Example 2

Growing Metarhizium anisopliae Strain 15013-1 in Liquid Culture at 2 LBenchtop Scale

A siliconized 250 mL shake flask containing 50 mL of cottonseed 10C: 1Nmedium was inoculated with M. anisopliae strain 15013-1 aerialconidiospores (AC) at a final concentration of 5×10⁶ AC/mL. The seedculture was grown at 28° C., 200 rpm for 2 days. After recording the MSand SS concentrations, 40 mL of the seed culture was inoculated into 1 Lof Soy 10C:1N (25% glucose+75% fructose) medium. During production, 10%NaOH was used to prevent the pH from falling under pH 5.5. Agitation wasstarted at 800 RPM, and was increased to maintain the DO above 30% witha maximum RPM of 1200. The temperature was 28° C. throughout thefermentation, and the length of the fermentation was 7 days. Variousparameters were monitored during the fermentation and include, but werenot limited to CER, DO, ammonia concentrations, pH, DCW, MS production,and SS production (FIGS. 2-5). At the end of fermentation, 1.368×10⁵MS/mL±0.046×10⁵ MS/mL, 7.000×10⁷ SS/mL±0.354×10⁷ SS/mL, and DCW of 24.8g/kg were produced in the culture.

Example 3

Growing Metarhizium anisolpliae Strain 15013-1 in Liquid CultureBioreactor without Feeding

A seed culture was started by inoculating 500 mL of cottonseed 10C:1Nmedium with Metarhizium anisopilae strain 15013-1 at a finalconcentration of 5×10⁶ AC/mL. The seed cultures were incubated at 180rpm and 28° C. for 2 days. A volume of 400 mL of the seed culture wasmixed with 600 mL of sterile water and inoculated into each tank tobring the final volume to 10 L of soy 10C (25% glucose+75% fructose):1Nmedium. The production culture pH was controlled at pH 5.5 with NaOH forbase addition and H₂SO₄ for acid addition. The pressure in the tank wasset at 1 barg and the agitation was allowed to go up to a maximum of 850RPM to maintain the dissolved oxygen of 20%. Various parameters weremonitored during the fermentation and include, but were not limited to,CER, DO, ammonia concentrations, pH, DCW, MS production, and SSproduction (FIGS. 6-9). At the end of fermentation, 9.433×10⁴MS/mL±1.366×10⁴ MS/mL, 2.750×10⁷ SS/mL±1.061×10⁷ SS/mL, and DCW of 25.0g/kg were produced in the culture.

Growing Metarhizium anisopliae Strain 15013-1 in Liquid CultureBioreactor with Carbon Source Feed

A seed culture was started by inoculating 500 mL of cottonseed 10C:1Nmedium with Metarhizium anisopilae strain 15013-1 at a finalconcentration of 5×10⁶ AC/mL. The seed cultures were incubated at 180rpm and 28° C. for 2 days. A volume of 400 mL of the seed culture wasmixed with 600 mL of sterile water and inoculated into each the tank tobring the final volume to 10 L of appropriate production medium. Theproduction culture pH was controlled at pH 5.5 with NaOH for baseaddition and H₂SO₄ for acid addition. The pressure in the tank was setat 1 barg and the agitation was allowed to go up to a maximum of 850 RPMto maintain the dissolved oxygen of 20%. At 88.5 h, water was added tothe tank in an attempt to loosen up the broth and clear some of thematerial on the head plate to allow for base to be added for pH control.A 50% (w/w) fructose solution was fed after the initial glucose andfructose was exhausted. Various parameters were monitored during thefermentation and include, but were not limited to, CER, DO, ammoniaconcentrations, pH, DCW, MS production, and SS production (FIGS. 10-13).At the end of fermentation, 1.928×10⁵ MS/mL±0.074×10⁵ MS/mL, 6.250×10⁶SS/mL±1.768×10⁶ SS/mL, and DCW of 38.1 g/kg were produced in theculture.

Growing Metarhizium anisopliae Strain 15013-1 to Enrich for SubmergedSpore Production in Liquid Culture Bioreactor

A seed culture was started by inoculating 500 mL of cottonseed 10C:1Nmedium with Metarhizium anisopilae strain 15013-1 at a finalconcentration of 5×10⁶ AC/mL. The seed culture was incubated at 100 rpmand 28° C. for 2 days. A volume of 400 mL of the seed culture was mixedwith 600 mL of sterile water and inoculated into each the tank to bringthe final volume to 10 L of soy 10C (25% glucose+75% fructose):1Nmedium. The production culture pH was controlled at pH 5.5 with NaOH forbase addition and H₂SO₄ for acid addition. The agitation was set at 350RPM and the dissolved oxygen was allowed to drop. This reduction inagitation created a stress condition, most likely a reduction inavailability of oxygen, which resulted in an increase in the productionof submerged spores during the production fermentation. Variousparameters were monitored during the run and include, but were notlimited to, CER (carbon dioxide evolution rate), OUR (oxygen uptakerate), DO (dissolved oxygen), pH, DCW (dry cell weight), microsclerotia(MS) concentration, and submerged spore (SS) concentration (FIGS.14-17). At the end of fermentation, 4.500×10³ MS/mL±1.146×10³ MS/mL,4.294×10⁸ SS/mL±0.186×10⁸ SS/mL, and DCW of 50.8 g/kg were produced inthe culture.

All publications, patents and patent applications mentioned in thespecification indicate the level of those skilled in the art to whichthis disclosure pertains. All publications, patents and patentapplications are incorporated by reference to the same extent as if eachindividual publication, patent or patent application was specificallyand individually indicated to be incorporated by reference.

Although the foregoing disclosure has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, certain changes and modifications may be practiced withinthe scope of the appended claims.

What is claimed is:
 1. A method of producing a fungal entomopathogenic product comprising: a. obtaining aerial conidiospores of a fungal entomopathogen selected from the group consisting of: Metarhizium anisopliae 15013-1, Metarhizium robertsii 23013-3, and Metarhizium anisopliae 3213-1; b. inoculating the aerial conidiospores into a liquid medium to generate a fungal entomopathogenic seed culture by fermentation; and c. inoculating the fungal entomopathogen seed culture into the liquid medium to generate a fungal entomopathogenic product by fermentation, wherein the liquid medium comprises a first carbon source, a second carbon source, and a nitrogen source.
 2. The method of claim 1, further comprising obtaining the fungal entomopathogenic product from the liquid medium.
 3. The method of claim 2, wherein obtaining the fungal entomopathogenic product from the liquid medium comprises vacuum drying.
 4. The method of claim 1, wherein the first carbon source is in a limiting concentration to facilitate a non-optimal or stress condition of the fungal entomopathogen, wherein the non-optimal or stress condition results in conversion of the fungal entomopathogen to microsclerotia.
 5. The method of claim 4, wherein the first carbon source comprises 10 to 35 percent by weight/volume of the total medium.
 6. The method of claim 1, wherein the second carbon source is in a non-limiting concentration.
 7. The method of claim 1, wherein the second carbon source creates a non-optimal or stress condition of the fungal entomopathogen, wherein the non-optimal or stress condition results in conversion of the fungal entomopathogen to microsclerotia.
 8. The method of claim 1, wherein the first carbon source comprises a glucose molecule.
 9. The method of claim 1, wherein the second carbon source comprises a fructose, a galactose, a sorbitol, a sorbose, a sucrose, an arabinose, a maltodextrin, a ribose, or a xylose molecule.
 10. The method of claim 1, further comprising adjusting a fermentation parameter to create a non-optimal or stress condition that results in a change of a physiological state of the fungal entomopathogen, wherein the non-optimal or stress condition results in conversion of the fungal entomopathogen to microsclerotia.
 11. The method of claim 1, wherein obtaining aerial conidospores of a fungal entomopathogen comprises first generating aerial conidiospores of the fungal entomopathogen on agar media or solid state media.
 12. The method of claim 1, wherein the fungal entomopathogenic product comprises a spore, a conidiospore, a blastospore, an aerial conidiospore, a submerged conidiospore, a submerged blastospore, and/or a microsclerotium. 